Viral therapeutics

ABSTRACT

A method for identifying a substance for affecting a viral infection is described. The method comprises providing a lipid globule targeting sequence, as a first component; providing a lipid globule, as a second component; contacting the two components with a substance to be tested under conditions that would permit the two components to interact in the absence of the substance; and determining whether the substance disrupts the interaction between the first and second components; where the targeting sequence comprises a hepatitis C virus (HCV) core protein or a fragment, derivative, variant or homologue thereof.

RELATED APPLICATIONS

[0001] This application is a Continuation-in-Part of U.S. applicationSer. No. 09/201,916 filed Dec. 1, 1998, which claims priority to GreatBritain patent application No. 9825951.8, filed Nov. 26, 1998, thedisclosures of which are herein incorporated by reference in theirentireties.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates to substances capable of modulating theinteraction between viral proteins capable of binding to intracellularlipid globules, cellular adipocyte-specific differentiation-relatedprotein and intracellular lipid globules. The invention also relates toassays for identifying such substances and the use of these substancesin affecting viral infection.

[0004] 2. Description of the Related Art

[0005] Hepatitis C virus (HCV) is a major causative agent of chronichepatitis and liver disease. It is estimated that, worldwide,approximately 300 million individuals are infected with the virus, 20%of whom are likely to develop mild to severe liver disease or carcinoma.Apart from the risk of succumbing to the long term effects of infection,these individuals also represent a large reservoir of virus for futuretransmissions. To date, the only widely used therapy for HCV istreatment with interferon. However, sustained response is achieved inonly about 20% of cases. Moreover, no vaccine currently exists toprotect against infection. Since growth of the virus has not beenpossible to date in tissue culture systems, very little is known alsoabout the molecular events which occur during viral replication.

[0006] The core protein of HCV is predicted to constitute the capsid ofvirus particles. From various studies, expression of this proteinresults in a range of effects on intracellular processes, including adecrease in transcription of genes from HBV and HIV and alterations toapoptosis. There is also evidence from a study on transgenic mice thatliver-specific expression of core may be linked to the development ofsteatosis (fatty liver), a condition commonly found in HCV-infectedindividuals which is characterized by the accumulation of fat depositswithin hepatocytes. Thus, core protein may also influence lipidmetabolism within the liver. Other results from studies on human serasuggest that HCV virus particles are found associated with lipoproteinparticles which are produced by the liver. It has also been shown thatHCV core protein associates with lipid droplets within cells (Barba etal., 1997; Moradpour et al., 1996). The droplets are storagecompartments for both triacylglycerols and cholesterol esters which canbe used as substrates for oxidation in mitochondria and for theformation of membranes. In specialized cells, stored cholesterol is usedfor steroid hormone synthesis.

[0007] Within the liver, lipid droplets also function as a site forstorage of precursors of the lipid which is secreted from this organ inthe form of lipoprotein particles. Although lipid droplets wereidentified several decades ago and they can be readily detected bystaining methods, very little is known about the processes of assembly,storage and disassembly within the cell.

SUMMARY OF THE INVENTION

[0008] We have surprisingly shown that expression of HCV core proteinand its resultant association with intracellular lipid droplets resultsin the loss of a protein, termed adipocyte-specificdifferentiation-related protein (ADRP), from the droplets. ADRP has beenfound to associate with lipid droplets in a range of cell types and incertain organs. It has been proposed that ADRP may be required formaintenance of lipid droplets within cells, however the precise functionof the protein has not been identified. Furthermore, progressiveincreases in core expression result in diminishing amounts of ADRP toundetectable levels. We have also identified regions of the HCV coreprotein which are required for both lipid globule association anddown-regulation of ADRP levels. Without wishing to be bound by theory,we believe that displacement of ADRP by HCV core protein may be a factorinvolved in HCV infection. Thus, the interaction of HCV core withintracellular lipid globules and associated displacement of ADRPrepresents a target for therapies designed to prevent or reduce theeffects and/or progression of HCV infection.

[0009] One embodiment of the present invention relates to a method foridentifying a substance capable of affecting a viral infection. Themethod comprises providing a lipid globule targeting sequence, as afirst component; providing a lipid globule, as a second component;contacting the two components with a substance to be tested underconditions that would permit the two components to interact in theabsence of the substance; and determining whether the substance disruptsthe interaction between the first and second components.

[0010] Preferably, the targeting sequence comprises a hepatitis C virus(HCV) core protein or a fragment thereof, or a GB virus-B core proteinor a fragment thereof. More preferably, the targeting sequence furthercomprises variants or homologues of the hepatitis C virus (HCV) coreprotein or the GB virus-B core protein.

[0011] In a variation, to the method, the substance to be tested isadministered to a cell, the lipid globule targeting sequence isexpressed in the cell, and the lipid globule is a natural constituent ofthe cell. The lipid globule targeting sequence may be naturally orrecombinantly expressed in the cell.

[0012] In a further variation to the method, a virus is administered toa cell in the absence of the substance which has been determined todisrupt the interaction between the first and second components. Thevirus is also administered to the cell in the presence of the substance.It is then determined if the substance reduces or abolishes thesusceptibility of the cell to viral infection or the effects of viralinfection. The cell is preferably a liver cell.

[0013] In another preferred mode of the invention, the lipid globuletargeting sequence comprises amino acids of the HCV core proteinselected from the group consisting of 125 to 144, 161 to 166 and thecombination thereof.

[0014] The viral infection is preferably a hepatitis infection or otherviral infection of the human or animal liver.

[0015] Another aspect of the invention relates to the substanceidentified by the above methods. Preferably, the substance has notpreviously been known to affect viral infection.

[0016] Another method for identifying a substance for treating orpreventing a viral infection, comprises: administering the substance toa mammalian cell; and identifying whether the administration of thesubstance upregulates expression of adipocyte-specific differentiationrelated protein (ADRP) in the mammalian cell.

[0017] Another aspect of the invention relates to a substance capable ofdisrupting an interaction between a lipid globule targeting sequence anda lipid globule for use in affecting a viral infection, wherein thetargeting sequence comprises a hepatitis C virus (HCV) core protein or afragment thereof, or a GB virus-B core protein or fragment thereof.

[0018] A further aspect of the invention relates to a polypeptidecomprising a lipid globule targeting sequence for use in preventing ortreating a viral infection, wherein the targeting sequence comprises anHCV core protein or a fragment, variant or homologue thereof, or a GBvirus-B core protein or a fragment, variant or homologue thereof.

[0019] Preferably, the polypeptide comprises the targeting sequencecomprising amino acids of the HCV core protein selected from the groupconsisting of 125 to 144, 161 to 166 and the combination thereof.

[0020] Another variation to the present invention relates to apharmaceutical composition comprising the polypeptide and apharmaceutically acceptable carrier or diluent. Further, a method isprovided for treating or preventing a viral infection comprisingadministering an effective amount of the pharmaceutical composition.

[0021] Another method for treating or preventing a viral infection isprovided, comprising administering an effective amount of apharmaceutical composition comprising the substance identified asdisrupting the interaction between the first and second components, anda pharmaceutically acceptable carrier or diluent.

[0022] A further method is provided for treating or preventing a viralinfection comprising administering an effective amount of apharmaceutical composition comprising the substance identified asupregulating expression of adipocyte-specific differentiation relatedprotein (ADRP) in a mammalian cell.

[0023] In accordance with another embodiment of the present invention, apolynucleotide is provided encoding a polypeptide comprising a lipidglobule targeting sequence for use in treating or preventing a viralinfection.

[0024] Another preferred embodiment of the present invention relates toa method for determining whether a test substance is capable of treatingor preventing a viral infection. The method comprises: providing a lipidglobule targeting sequence, as a first component, the targeting sequencecomprising a hepatitis C virus (HCV) core protein or a fragment thereof,or a GB virus-B core protein or fragment thereof; providing a lipidglobule, as a second component; incubating the first and secondcomponents with the test substance under conditions that would permitthe first and second components to interact with one another in theabsence of the test substance; and determining whether the testsubstance disrupts the interaction between the first and secondcomponents.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1 shows Western blots probed with antibodies to HCV coreprotein.

[0026]FIG. 2 shows confocal microscopy images of the intracellularlocalization of core proteins and lipid droplets.

[0027]FIG. 3 shows confocal microscopy images of cells illustrating theeffect of expression of HCV core proteins on the ability to detect ADRPin BHKC13 cells.

[0028]FIG. 4 shows confocal microscopy images of cells illustrating theeffect of expression of HCV core protein on the abundance of ADRP.

[0029]FIG. 5 shows Western blots probed with antibodies to HCV coreprotein and adipophilin.

[0030]FIG. 6 shows Western blots probed with antibodies to HCV coreprotein.

[0031]FIG. 7 shows the amino acid sequence comparison between thepredicted core proteins of HCV and GBV-B.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0032] Although in general the techniques mentioned herein are wellknown in the art, reference may be made in particular to Sambrook etal., Molecular Cloning, A Laboratory Manual (1989) and Ausubel et al.,Current Protocols in Molecular Biology (1995), John Wiley & Sons, Inc.

[0033] A. Proteins/Polypeptides

[0034] The term “protein” includes single-chain polypeptide molecules aswell as multiple-polypeptide complexes where individual constituentpolypeptides are linked by covalent or non-covalent means. The term“polypeptide” includes peptides of two or more amino acids in length,typically having more than 5, 10 or 20 amino acids.

[0035] 1. Lipid Globule Targeting Sequences

[0036] The term “lipid globule targeting sequence” means an amino acidsequence which is capable of association with a lipid globule,preferably a biologically occurring lipid globule such as anintracellular lipid globule as found in adipocytes or a secreted lipidglobule as found in mammalian milk. In addition, the lipid globuletargeting sequence is preferably capable of association with a lipidglobule when linked to a protein of interest such that the protein ofinterest is also associated with the lipid globule by virtue of beinglinked to the targeting sequence. Lipid globule association may takeplace within a non-cellular and/or extra-cellular environment, such asin an apparatus—for example a tube or vat. Alternatively, it may takeplace in a cellular environment where the expressed targeting sequenceis directed to intracellular lipid droplets or the membranes of suchdroplets.

[0037] The ability of an amino acid sequence to associate with/targetlipid globules can be assessed either in vitro or in vivo. For example,a candidate targeting sequence may be added to a dispersion of lipidglobules (such as a mixture of phospholipid and triacylglycerol) in anaqueous solvent, the mixture sonicated and the degree of partitionbetween aqueous and lipid phases determined by fractionation. Typicallyfractionation of the mixture would involve increasing the density of thesolution with sorbitol or sodium bromide and ultracentrifuging thesolution. The lipid complexes migrate to the top of the centrifuge tubeand this upper lipid layer is then examined for candidate targetingsequence. Preferably, a suitable lipid globule targeting sequence shouldpartition at least 50:50 lipid:aqueous phase, more preferably at least75:25, 80:20 or 90:10.

[0038] Another suitable test may involve introducing a polynucleotideencoding a candidate sequence, optionally linked to a protein ofinterest, into a milk-producing cell in culture and determining whether,the targeting sequence/protein of interest has been secreted into theculture medium. The immunocytochemical technique illustrated in theExamples may also be used.

[0039] Lipid globule targeting sequences used according to the presentinvention are also preferably capable of displacing ADRP fromintracellular lipid globules and/or of reducing the levels of ADRPprotein in a cell when expressed in, or administered to, the cell (asillustrated for HCV core protein in the Examples). Suitable techniquesfor determining whether a candidate sequence has these properties aredescribed below.

[0040] Suitable lipid globule targeting sequences may be obtained froman HCV core protein. The amino acid sequence of the HCV core protein hasbeen obtained for a large number of different HCV isolates. Thesesequences are readily available to the skilled person. One suchsequence, for HCV strain Glasgow, is set out in SEQ ID No. 1. The meansfor cloning and identifying new HCV strains, and thus obtaining furthercore sequences, are described in EP-B-318,216.

[0041] According to the present invention, it is preferred to usefragments of the HCV core protein which are capable of targetingmolecules, to which they are linked, to lipid globules. Amino acidnumbering for preferred fragments set out below is with reference to SEQID. No. 1. However it will be understood that equivalent fragments ofthe core protein of other HCV strains/isolates may also be used. An HCVcore protein-derivable lipid globule targeting sequence of the inventionis preferably a minimal amino acid sequence which can target a molecule,typically a protein, to lipid globules. The minimal sequence willtypically comprise a hydrophobic amino acid sequence derived from aminoacids 120 to 169 of an HCV core sequence, preferably linked to ahydrophilic amino acid sequence of at least 8, preferably 10, morepreferably at least 12 amino acids. It is not necessary for thehydrophilic sequence to be contiguous with the hydrophobic sequence. Forexample, a protein of interest may be placed between the two sequencessuch that the hydrophilic sequence is at the N-terminus and thehydrophobic sequence is at the C-terminus.

[0042] The hydrophobic amino acid sequence typically comprises at least10, preferably at least 15 or 20 contiguous amino acids and has ahydropathy index of at least +40 kJ/mol (determined, for example,theoretically as described by Engelman et al., 1986. The hydrophilicamino acid sequence typically has a hydropathy plot of less than −20kJ/mol, preferably less than −40 kJ/mol.

[0043] Preferred HCV core fragments contain amino acids 161 to 166 (SEQID. No. 3). It is also preferred to use fragments of the HCV coreprotein that contain amino acids 125 to 144 (SEQ ID. No. 2). In apreferred embodiment, HCV core protein fragments of the inventioncontain both amino acids 125 to 144 and amino acids 161 to 166. In anespecially preferred embodiment, the lipid targeting sequence of theinvention comprises a hydrophilic amino acid sequence containing aminoacids 1 to 8 of the HCV core sequence. Other preferred fragments containamino acids 1 to 173 or 1 to 169.

[0044] Since it has also now been shown that amino acids 9 to 43, 49 to75, 80 to 118 and 155 to 161 are not required for lipid association,preferred HCV core protein fragments of the invention lack one or moreof these sequences. Suitable fragments will be at least about 5, e.g.10, 12, 15 or 20 amino acids in size and preferably have less than 100,90, 80, 70, 60 or 50 amino acids. In a preferred aspect, fragmentscontain an HCV epitope.

[0045] Lipid globule targeting sequences of the invention, for exampleHCV core protein sequences and fragments thereof, may, however, be partof a larger polypeptide, for example a fusion protein. In this case, theadditional polypeptide sequences are preferably polypeptide sequenceswith which the lipid globule targeting sequence of the invention is notnormally associated.

[0046] It will be understood that lipid globule targeting sequences ofthe invention are not limited to sequences obtained from HCV coreprotein but also include homologous sequences obtained from any source,for example related viral proteins, cellular homologues and syntheticpeptides, as well as variants or derivatives thereof. Thus, the presentinvention covers variants, homologues or derivatives of the targetingsequences of the present invention, as well as variants, homologues orderivatives of the nucleotide sequence coding for the targetingsequences of the present invention.

[0047] In the context of the present invention, a homologous sequence istaken to include an amino acid sequence which is at least 60, 70, 80 or90% identical, preferably at least 95 or 98% identical at the amino acidlevel over at least 5, preferably 8, 10, 15, 20, 30 or 40 amino acidswith an HCV core protein lipid targeting sequence, for example as shownin the sequence listing herein. In particular, homology should typicallybe considered with respect to those regions of the targeting sequenceknown to be essential for lipid globule association rather thannon-essential neighboring sequences. Homology comparisons can beconducted by eye, or more usually, with the aid of readily availablesequence comparison programs. These commercially available computerprograms can calculate % homology between two or more sequences. Atypical example of such a computer program is CLUSTAL.

[0048] Sequence homology (or identity) may moreover be determined usingany suitable homology algorithm, using for example default parameters.Advantageously, the BLAST algorithm is employed, with parameters set todefault values. The BLAST algorithm is described in detail athttp://www.ncbi.nih.gov/BLAST/blast_help.html, which is incorporatedherein by reference. The search parameters are defined as follows, andare advantageously set to the defined default parameters.

[0049] Advantageously, “substantial homology” when assessed by BLASTequates to sequences which match with an EXPECT value of at least about7, preferably at least about 9 and most preferably 10 or more. Thedefault threshold for EXPECT in BLAST searching is usually 10.

[0050] BLAST (Basic Local Alignment Search Tool) is the heuristic searchalgorithm employed by the programs blastp, blastn, blastx, tblastn, andtblastx; these programs ascribe significance to their findings using thestatistical methods of Karlin and Altschul (seehttp://www.ncbi.nih.gov/BLAST/blast help.html) with a few enhancements.The BLAST programs were tailored for sequence similarity searching, forexample to identify homologues to a query sequence. The programs are notgenerally useful for motif-style searching. For a discussion of basicissues in similarity searching of sequence databases, see Altschul etal., 1994.

[0051] The five BLAST programs available at http://www.ncbi.nlm.nih.govperform the following tasks:

[0052] blastp—compares an amino acid query sequence against a proteinsequence database;

[0053] blastn—compares a nucleotide query sequence against a nucleotidesequence database;

[0054] blastx—compares the six-frame conceptual translation products ofa nucleotide query sequence (both strands) against a protein sequencedatabase;

[0055] tblastn—compares a protein query sequence against a nucleotidesequence database dynamically translated in all six reading frames (bothstrands).

[0056] tblastx—compares the six-frame translations of a nucleotide querysequence against the six-frame translations of a nucleotide sequencedatabase.

[0057] BLAST uses the following search parameters:

[0058] HISTOGRAM—Display a histogram of scores for each search; defaultis yes. (See parameter H in the BLAST Manual).

[0059] DESCRIPTIONS—Restricts the number of short descriptions ofmatching sequences reported to the number specified; default limit is100 descriptions. (See parameter V in the manual page). See also EXPECTand CUTOFF.

[0060] ALIGNMENTS—Restricts database sequences to the number specifiedfor which high-scoring segment pairs (HSPs) are reported; the defaultlimit is 50. If more database sequences than this happen to satisfy thestatistical significance threshold for reporting (see EXPECT and CUTOFFbelow), only the matches ascribed the greatest statistical significanceare reported. (See parameter B in the BLAST Manual).

[0061] EXPECT—The statistical significance threshold for reportingmatches against database sequences; the default value is 10, such that10 matches are expected to be found merely by chance, according to thestochastic model of Karlin and Altschul (1990). If the statisticalsignificance ascribed to a match is greater than the EXPECT threshold,the match will not be reported. Lower EXPECT thresholds are morestringent, leading to fewer chance matches being reported. Fractionalvalues are acceptable. (See parameter E in the BLAST Manual).

[0062] CUTOFF—Cutoff score for reporting high-scoring segment pairs(HSPs). The default value is calculated from the EXPECT value (seeabove). HSPs are reported for a database sequence only if thestatistical significance ascribed to them is at least as high as wouldbe ascribed to a lone HSP having a score equal to the CUTOFF value.Higher CUTOFF values are more stringent, leading to fewer chance matchesbeing reported. (See parameter S in the BLAST Manual). Typically,significance thresholds can be more intuitively managed using EXPECT.

[0063] MATRIX—Specify an alternate scoring matrix for BLASTP, BLASTX,TBLASTN and TBLASTX. The default matrix is BLOSUM62 (Henikoff &Henikoff, 1992). The valid alternative choices include: PAM40, PAM120,PAM250 and IDENTITY. No alternate scoring matrices are available forBLASTN; specifying the MATRIX directive in BLASTN requests returns anerror response.

[0064] STRAND—Restrict a TBLASTN search to just the top or bottom strandof the database sequences; or restrict a BLASTN, BLASTX or TBLASTXsearch to just reading frames on the top or bottom strand of the querysequence.

[0065] FILTER—Mask off segments of the query sequence that have lowcompositional complexity, as determined by the SEG program of Wootton &Federhen (1993), or segments consisting of short-periodicity internalrepeats, as determined by the XNU program of Clayerie & States (1993),or, for BLASTN, by the DUST program of Tatusov and Lipman (seehttp://www.ncbi.nlm.nih.gov). Filtering can eliminate statisticallysignificant but biologically uninteresting reports from the blast output(e.g. hits against common acidic-, basic- or proline-rich regions),leaving the more biologically interesting regions of the query sequenceavailable for specific matching against database sequences.

[0066] Low complexity sequence found by a filter program is substitutedusing the letter “N” in nucleotide sequence (e.g., “NNNNNNNNNNNNN”) andthe letter “X” in protein sequences (e.g., “XXXXXXXXX”).

[0067] Filtering is only applied to the query sequence (or itstranslation products), not to database sequences. Default filtering isDUST for BLASTN, SEG for other programs.

[0068] It is not unusual for nothing at all to be masked by SEG, XNU, orboth, when applied to sequences in SWISS-PROT, so filtering should notbe expected to always yield an effect. Furthermore, in some cases,sequences are masked in their entirety, indicating that the statisticalsignificance of any matches reported against the unfiltered querysequence should be suspect.

[0069] NCBI-gi Causes NCBI gi identifiers to be shown in the output, inaddition to the accession and/or locus name.

[0070] Most preferably, sequence comparisons are conducted using thesimple BLAST search algorithm provided athttp://www.ncbi.nlm.nih.gov/BLAST.

[0071] Other computer program methods to determine identify andsimilarity between the two sequences include but are not limited to theGCG program package (Devereux et al 1984) and FASTA (Altschul et al.1990).

[0072] Lipid globule targeting sequences of the invention, for exampleHCV core protein sequences, variants, homologues and fragments thereof,may be modified for use in the present invention. Typically,modifications are made that maintain the hydrophobicity/hydrophilicityof the sequence Amino acid substitutions may be made, for example from1, 2 or 3 to 10, 20 or 30 substitutions provided that the modifiedsequence retains the ability to target molecules to lipid globules.Amino acid substitutions may include the use of non-naturally occurringanalogues, for example to increase blood plasma half-life of atherapeutically administered polypeptide.

[0073] Conservative substitutions may be made, for example according tothe Table below. Amino acids in the same block in the second colunm andpreferably in the same line in the third column may be substituted foreach other: ALIPHATIC Non-polar G A P I L V Polar - uncharged C S T M NQ Polar - charged D E K R AROMATIC H F W Y

[0074] The terms “variant”, “homologue” or “derivative” in relation tothe targeting sequence of the present invention include any substitutionof, variation of, modification of, replacement of, deletion of oraddition of one (or more) amino acids from or to the sequence providingthe resultant amino acid sequence has a lipid globule targetingactivity, preferably having at least the same activity of the targetingsequences presented in the sequence listings.

[0075] Proteins of the invention comprising lipid globule targetingsequences are typically made by recombinant means, for example asdescribed below. However they may also be made by synthetic means usingtechniques well known to skilled persons such as solid phase synthesis.Proteins of the invention may also be produced as fusion proteins, forexample to aid in extraction and purification. Examples of fusionprotein partners include glutathione-S-transferase (GST), 6×His, GAL4(DNA binding and/or transcriptional activation domains) andβ-galactosidase. It may also be convenient to include a proteolyticcleavage site between the fusion protein partner and the HCV coreprotein sequence and/or between the HCV core protein sequence and theprotein of interest to allow removal of fusion protein sequences.Preferably the fusion protein will not hinder the lipid targeting effectof the lipid globule targeting sequence. The targeting sequence may belinked to either the N-terminus or the C-terminus of the fusion proteinpartners or proteins of interest

[0076] Proteins of the invention may be in a substantially isolatedform. It will be understood that the protein may be mixed with carriersor diluents which will not interfere with the intended purpose of theprotein and still be regarded as substantially isolated. A protein ofthe invention may also be in a substantially purified form, in whichcase it will generally comprise the protein in a preparation in whichmore than 90%, e.g. 95%, 98% or 99% of the protein in the preparation isa protein of the invention.

[0077] 2. Adipocyte-specific Differentiation Related Protein

[0078] In another embodiment of the in vitro assay methods of thepresent invention, ADRP may be added to the mixture to compete with alipid globule targeting sequence. ADRP protein (also known asadipophilin) can be obtained in a number of ways, for example bypurification from mammalian milk or mammalian cell lines (Heid et al.,1998). Alternatively, ADRP can be obtained by recombinant means, asdescribed above for lipid globule targeting sequences. The nucleotidesequence of human ADRP, for example, is given in U.S. Pat. No. 5,739,009and shown in SEQ ID. No. 4. The polypeptide sequence of human ADRP isshown in SEQ ID No. 5. Since ADRP is capable of binding lipid globules,it can also be considered to be a lipid globule targeting sequencewithin the scope of the invention. Consequently, the disclosure aboveand below relating to lipid globule targeting sequences also generallyapplies, where appropriate, to ADRP sequences, for example the use offragments, homologues, derivatives and variants. In particular, ADRPsequences may be modified for use in the assays of the present inventionas described above.

[0079] B. Polynucleotides and Vectors.

[0080] Polynucleotides of the invention comprise nucleic acid sequencesencoding the lipid globule targeting sequences of the invention. It willbe understood by a skilled person that numerous differentpolynucleotides can encode the same polypeptide as a result of thedegeneracy of the genetic code. In addition, it is to be understood thatskilled persons may, using routine techniques, make nucleotidesubstitutions that do not affect the polypeptide sequence encoded by thepolynucleotides of the invention to reflect the codon usage of anyparticular host organism in which the polypeptides of the invention areto be expressed.

[0081] Polynucleotides of the invention may comprise DNA or RNA. Theymay be single-stranded or double-stranded. They may also bepolynucleotides which include within them synthetic or modifiednucleotides. A number of different types of modification tooligonucleotides are known in the art. These include methylphosphonateand phosphorothioate backbones, addition of acridine or polylysinechains at the 3′ and/or 5′ ends of the molecule. For the purposes of thepresent invention, it is to be understood that the polynucleotidesdescribed herein may be modified by any method available in the art.Such modifications may be carried out in order to enhance the in vivoactivity or life span of polynucleotides of the invention.

[0082] The terms “variant”, “homologue” or “derivative” in relation tothe nucleotide sequence coding for the lipid targeting sequence of thepresent invention include any substitution of, variation of,modification of, replacement of, deletion of or addition of one (ormore) nucleic acid from or to the sequence providing the resultantnucleotide sequence codes for a protein having lipid targeting activity,preferably having at least the same activity of the targeting sequencespresented in the sequence listings.

[0083] As indicated above, with respect to sequence homology, preferablythere is at least 75%, more preferably at least 85%, more preferably atleast 90% homology to the sequences shown in the sequence listingherein. More preferably there is at least 95%, more preferably at least98%, homology. Nucleotide homology comparisons may be conducted asdescribed above.

[0084] The present invention also encompasses nucleotide sequences thatare capable of hybridizing selectively to the sequences presentedherein, or any variant, fragment or derivative thereof, or to thecomplement of any of the above. Nucleotide sequences are preferably atleast 15 nucleotides in length, more preferably at least 20, 30, 40 or50 nucleotides in length.

[0085] The term “hybridization” as used herein shall include “theprocess by which a strand of nucleic acid joins with a complementarystrand through base pairing” (Coombs J (1994) Dictionary ofBiotechnology, Stockton Press, New York N.Y.) as well as the process ofamplification as carried out in polymerase chain reaction technologiesas described in Dieffenbach C W and G S Dveksler (1995, PCR Primer, aLaboratory Manual, Cold Spring Harbor Press, Plainview N.Y.).

[0086] Polynucleotides of the invention capable of selectivelyhybridizing to the nucleotide sequences presented herein, or to theircomplement, will be generally at least 70%, preferably at least 80 or90% and more preferably at least 95% or 98% homologous to thecorresponding nucleotide sequences presented herein over a region of atleast 20, preferably at least 25 or 30, for instance at least 40, 60 or100 or more contiguous nucleotides. Preferred polynucleotides of theinvention will comprise regions homologous to nucleotides 715 to 774and/or nucleotides 826 to 840 of SEQ ID No. 1, preferably at least 80 or90% and more preferably at least 95% homologous to nucleotides 715 to774 and/or nucleotides 826 to 840 of SEQ ID No. 1.

[0087] The term “selectively hybridizable” means that the polynucleotideused as a probe is used under conditions where a target polynucleotideof the invention is found to hybridize to the probe at a levelsignificantly above background. The background hybridization may occurbecause of other polynucleotides present, for example, in the cDNA orgenomic DNA library being screening. In this event, background implies alevel of signal generated by interaction between the probe and anon-specific DNA member of the library which is less than 10 fold,preferably less than 100 fold as intense as the specific interactionobserved with the target DNA. The intensity of interaction may bemeasured, for example, by radiolabeling the probe, e.g. with ³²P.

[0088] Hybridization conditions are based on the melting temperature(Tm) of the nucleic acid binding complex, as taught in Berger and Kimmel(1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol152, Academic Press, San Diego Calif.), and confer a defined“stringency” as explained below.

[0089] Maximum stringency typically occurs at about Tm-5° C. (5° C.below the Tm of the probe); high stringency at about 5° C. to 10° C.below Tm; intermediate stringency at about 10° C. to 20° C. below Tm;and low stringency at about 20° C. to 25° C. below Tm. As will beunderstood by those of skill in the art, a maximum stringencyhybridization can be used to identify or detect identical polynucleotidesequences while an intermediate (or low) stringency hybridization can beused to identify or detect similar or related polynucleotide sequences.

[0090] In a preferred aspect, the present invention covers nucleotidesequences that can hybridize to the nucleotide sequence of the presentinvention under stringent conditions (e.g. 65° C. and 0.1×SSC{1×SSC=0.15M NaCl, 0.015 M Na₃ citrate pH 7.0).

[0091] Where the polynucleotide of the invention is double-stranded,both strands of the duplex, either individually or in combination, areencompassed by the present invention. Where the polynucleotide issingle-stranded, it is to be understood that the complementary sequenceof that polynucleotide is also included within the scope of the presentinvention.

[0092] Polynucleotides which are not 100% homologous to the sequences ofthe present invention but fall within the scope of the invention can beobtained in a number of ways. Other HCV core protein variants of the HCVcore protein sequence described herein may be obtained for example byprobing DNA libraries made from a range of HCV infected individuals, forexample individuals from different populations. In addition, otherviral, or cellular homologues particularly cellular homologues found inmammalian cells (e.g. rat, mouse, bovine and primate cells), may beobtained and such homologues and fragments thereof in general will becapable of selectively hybridizing to the sequences shown in thesequence listing herein. Such sequences may be obtained by probing cDNAlibraries made from or genomic DNA libraries from other animal species,and probing such libraries with probes comprising all or part of SEQ ID.1 under conditions of medium to high stringency. Similar considerationsapply to obtaining species homologues and allelic variants of ADRP.

[0093] Variants and strain/species homologues may also be obtained usingdegenerate PCR which will use primers designed to target sequenceswithin the variants and homologues encoding conserved amino acidsequences within the lipid globule targeting sequences of the presentinvention. Conserved sequences can be predicted, for example, byaligning the HCV core protein amino acid sequences from several HCVisolates. Such HCV sequence comparisons are widely available in the art.The primers will contain one or more degenerate positions and will beused at stringency conditions lower than those used for cloningsequences with single sequence primers against known sequences.

[0094] Alternatively, such polynucleotides may be obtained by sitedirected mutagenesis of characterized lipid globule targeting sequences,such as SEQ ID. No 1. This may be useful where for example silent codonchanges are required to sequences to optimize codon preferences for aparticular host cell in which the polynucleotide sequences are beingexpressed. Other sequence changes may be desired in order to introducerestriction enzyme recognition sites, or to alter the property orfunction of the polypeptides encoded by the polynucleotides.

[0095] Polynucleotides of the invention may be used to produce a primer,e.g. a PCR primer, a primer for an alternative amplification reaction, aprobe e.g. labeled with a revealing label by conventional means usingradioactive or non-radioactive labels, or the polynucleotides may becloned into vectors. Such primers, probes and other fragments will be atleast 15, preferably at least 20, for example at least 25, 30 or 40nucleotides in length, and are also encompassed by the termpolynucleotides of the invention as used herein.

[0096] Polynucleotides such as a DNA polynucleotides and probesaccording to the invention may be produced recombinantly, synthetically,or by any means available to those of skill in the art. They may also becloned by standard techniques.

[0097] In general, primers will be produced by synthetic means,involving a step wise manufacture of the desired nucleic acid sequenceone nucleotide at a time. Techniques for accomplishing this usingautomated techniques are readily available in the art.

[0098] Longer polynucleotides will generally be produced usingrecombinant means, for example using a PCR polymerase chain reaction)cloning techniques. This will involve making a pair of primers (e.g. ofabout 15 to 30 nucleotides) flanking a region of the lipid targetingsequence which it is desired to clone, bringing the primers into contactwith mRNA or cDNA obtained from an animal or human cell, performing apolymerase chain reaction under conditions which bring aboutamplification of the desired region, isolating the amplified fragment(e.g. by purifying the reaction mixture on an agarose gel) andrecovering the amplified DNA. The primers may be designed to containsuitable restriction enzyme recognition sites so that the amplified DNAcan be cloned into a suitable cloning vector

[0099] Polynucleotides of the invention can be incorporated into arecombinant replicable vector. The vector may be used to replicate thenucleic acid in a compatible host cell. Thus in a further embodiment,the invention provides a method of making polynucleotides of theinvention by introducing a polynucleotide of the invention into areplicable vector, introducing the vector into a compatible host cell,and growing the host cell under conditions which bring about replicationof the vector. The vector may be recovered from the host cell. Suitablehost cells include bacteria such as E. coli, yeast, mammalian cell linesand other eukaryotic cell lines, for example insect Sf9 cells.

[0100] Preferably, a polynucleotide of the invention in a vector isoperably linked to a control sequence that is capable of providing forthe expression of the coding sequence by the host cell, i.e. the vectoris an expression vector. The term “operably linked” means that thecomponents described are in a relationship permitting them to functionin their intended manner. A regulatory sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under condition compatible with the controlsequences.

[0101] A particularly preferred vector for use in the present inventioncomprises the control sequences naturally associated with ADRP genes,such as the human ADRP gene. Typically, the control sequences areoperably linked to a reporter gene. Such a vector may be used in theassays described below for identifying modulators of ADRP expression.The control sequences may be modified, for example by the addition offurther transcriptional regulatory elements to make the level oftranscription directed by the control sequences more responsive totranscriptional modulators.

[0102] Vectors of the invention may be transformed or transfected into asuitable host cell as described below to provide for expression of aprotein of the invention. This process may comprise culturing a hostcell transformed with an expression vector as described above underconditions to provide for expression by the vector of a coding sequenceencoding the protein, and optionally recovering the expressed protein.Alternatively, vectors comprising the control sequences naturallyassociated with ADRP genes operably linked to a reporter gene as areporter construct may be transformed or transfected into a host cell,for example for use in assays of the invention which measure the effectof candidate substances on transcription from the reporter construct ina cellular environment.

[0103] The vectors may be for example, plasmid or virus vectors providedwith an origin of replication, optionally a promoter for the expressionof the said polynucleotide and optionally a regulator of the promoter.The vectors may contain one or more selectable marker genes, for examplean ampicillin resistance gene in the case of a bacterial plasmid or aneomycin resistance gene for a mammalian vector. Vectors may be used,for example, to transfect or transform a host cell.

[0104] Control sequences operably linked to sequences encoding theprotein of the invention include promoters/enhancers and otherexpression regulation signals. These control sequences may be selectedto be compatible with the host cell for which the expression vector isdesigned to be used in. The term promoter is well-known in the art andencompasses nucleic acid regions ranging in size and complexity fromminimal promoters to promoters including upstream elements andenhancers.

[0105] The promoter is typically selected from promoters which arefunctional in mammalian, cells, although prokaryotic promoters andpromoters functional in other eukaryotic cells may be used. The promoteris typically derived from promoter sequences of viral or eukaryoticgenes. For example, it may be a promoter derived from the genome of acell in which expression is to occur. With respect to eukaryoticpromoters, they may be promoters that function in a ubiquitous manner(such as promoters of α-actin, β-actin, tubulin) or, alternatively, atissue-specific manner (such as promoters of the genes for pyruvatekinase). Tissue-specific promoters specific for liver cells areparticularly preferred, for example hepatitis B viral promoters,apoliprotein AII promoters human serum amyloid P component promoters orhuman protein C gene promoters. They may also be promoters that respondto specific stimuli, for example promoters that bind steroid hormonereceptors. Viral promoters may also be used, for example the Moloneymurine leukemia virus long terminal repeat (MMLV LTR) promoter, the roussarcoma virus (RSV) LTR promoter or the human cytomegalovirus (CMV) IEpromoter. As discussed above ADRP promoters are particularly preferredfor use in reporter constructs.

[0106] It may also be advantageous for the promoters to be inducible sothat the levels of expression of the heterologous gene can be regulatedduring the life-time of the cell. Inducible means that the levels ofexpression obtained using the promoter can be regulated.

[0107] In addition, any of these promoters may be modified by theaddition of further regulatory sequences, for example enhancersequences. Chimeric promoters may also be used comprising sequenceelements from two or more different promoters described above.

[0108] C. Host cells

[0109] Vectors and polynucleotides of the invention (and reporter geneconstructs described below) may be introduced into host cells for thepurpose of replicating the vectors/polynucleotides and/or expressing theproteins of the invention encoded by the polynucleotides of theinvention. Although the proteins of the invention may be produced usingprokaryotic cells as host cells, it is preferred to use eukaryoticcells, for example yeast, insect or mammalian cells, in particularmammalian cells. Particularly preferred cells are those with substantialamounts of intracellular lipid droplets/globules, for exampleadipocytes. Cells which are targeted by a particular virus which it isdesired to treat, for example liver cells, are also preferred.

[0110] Vectors/polynucleotides of the invention may introduced intosuitable host cells using a variety of techniques known in the art, suchas transfection, transformation and electroporation. Wherevectors/polynucleotides of the invention are to be administered toanimals, several techniques are known in the art, for example infectionwith recombinant viral vectors such as herpes simplex viruses andadenoviruses, direct injection of nucleic acids and biolistictransformation.

[0111] D. Protein Expression and Purification

[0112] Host cells comprising polynucleotides of the invention may beused to express proteins of the invention. Host cells may be culturedunder suitable conditions which allow expression of the proteins of theinvention. Expression of the proteins of the invention may beconstitutive such that they are continually produced, or inducible,requiring a stimulus to initiate expression. In the case of inducibleexpression, protein production can be initiated when required by, forexample, addition of an inducer substance to the culture medium, forexample dexamethasone or IPTG.

[0113] Proteins of the invention can be extracted from host cells by avariety of techniques known in the art, including enzymatic, chemicaland/or osmotic lysis and physical disruption. Although a large number ofdifferent purification protocols may be used, given the ability of theHCV core proteins of the invention to target proteins of interest tolipid globules, a preferred extraction/purification protocol involvescentrifuging cell homogenates at high speed (for example 100,000 g for60 mins at 2 to 4° C.) and removing the resulting layer of floatinglipids. This will function as a primary purification step. Furtherpurification can then be performed if necessary using, for example,column chromatography such as ion-exchange or affinity chromatography.Cells which secrete lipid globules may also conveniently be used and thelipid globules harvested from the culture supernatant.

[0114] Proteins associated with the membrane surrounding fat globulescan be fractionated into soluble and insoluble fractions by extractionwith 1% (w/v) Triton X-100/1.5 M NaCl/10 mM Tris (pH 7.0), by extractionwith 1.5% (w/v) dodecyl β-D maltoside/0.75 M aminohexanoic acid/10 mMHepes (pH 7.0) or by sequential extraction with these twodetergent-containing solutions (Patton and Huston, 1986). Suspension ofthe fat globule components in the detergent-containing solution can beachieved by using an all-glass homogenizer, and keeping on ice for 30 to60 min, after which insoluble and soluble materials can be separated bycentrifugation for 60 min at 2° C. and 150,000 g. The above conditionscan be modified to analyze whether core protein or a fusion proteincontaining core as a component is attached to fat globules. Otherdetergents, both ionic and non-ionic, along with salt solutions atvarious concentrations could be used to derive the proteinaceousmaterial from fat globules. The incubation times and temperatures may beoptimized by empirical means.

[0115] E. Assays

[0116] 1. Assays for Substances that Disrupt the Interaction Between aLipid Globule Targeting Sequence and a Lipid Globule

[0117] i. Candidate Substances

[0118] A substance which disrupts an interaction between the lipidglobule targeting sequence and lipid globule may do so in several ways.It may directly disrupt the binding of the lipid globule targetingsequence to the lipid globule by, for example, binding to the lipidglobule targeting sequence and masking or altering the site ofinteraction with the lipid globule. Alternatively, the candidatesubstance may compete for binding sites on the lipid globule surface anddisplace the lipid globule targeting sequence. Candidate substances ofthese types may conveniently be screened by in vitro binding assays as,for example, described below. Candidate substances may also be screenedusing an “in vivo” whole cell assay as described below. The term ‘invivo’ is intended to encompass experiments with cells in culture as wellas experiments with intact multicellular organisms.

[0119] A substance which can bind directly to the lipid globuletargeting sequence may also inhibit an interaction between the lipidglobule targeting sequence and the lipid globule by altering thesubcellular localization of the lipid globule targeting sequence thuspreventing the two components from coming into contact within the cell.This can also be tested in vivo using, for example the in vivo assaysdescribed below.

[0120] Alternatively, instead of preventing the association of thecomponents directly, the substance may suppress or enhance thebiologically available amount of lipid globule targeting sequence (forexample a viral protein component). This may be by inhibiting expressionof the viral protein comprising the lipid globule targeting sequence,for example at the level of transcription, transcript stability,translation, post-translational processing or post-translationalstability. An example of such a substance would be antisense RNA whichsuppresses the amount of HCV core protein mRNA translated into protein.

[0121] Suitable candidate substances include viral peptides, which insome cases may especially be of from about 5 to 20 amino acids in size,based on, for example, lipid globule targeting motifs found within theHCV core protein, or variants of such peptides in which one or moreresidues have been substituted. Peptide fragments of ADRP may also beused, including modified variants thereof. Peptides from panels ofpeptides comprising random sequences or sequences which have been variedconsistently to provide a maximally diverse panel of peptides may beused.

[0122] Suitable candidate substances also include antibody products (forexample, monoclonal and polyclonal antibodies, single chain antibodies,chimeric antibodies and CDR-grafted antibodies) which are specific forthe lipid globule targeting sequence or surface constituents of thelipid globules. Furthermore, combinatorial libraries, peptide andpeptide mimetics, defined chemical entities such as organic andinorganic compounds, oligonucleotides, and natural product libraries maybe screened for activity as inhibitors of an interaction between thelipid globule targeting sequence and a lipid globule in assays such asthose described below. The candidate substances may be used in aninitial screen in batches of, for example 10 substances per reaction,and the substances of those batches which show inhibition testedindividually. Candidate substances which show activity in in vitroscreens such as those described below can then be tested in in vivosystems, such as mammalian cells which will be exposed to the inhibitorand tested, for example, for susceptibility to viral infection.

[0123] ii. Assays

[0124] The assays of the invention may be in vitro assays or in vivoassays, for example using cell lines or an animal model, such as mice orchimpanzees.

[0125] In vitro Assay Systems

[0126] One type of in vitro assay for identifying substances whichdisrupt an interaction between the lipid globule targeting sequence anda lipid globule involves measuring in the presence or absence of acandidate substance the association of a targeting sequence with lipidglobules formed in vitro. Binding of targeting sequences to lipidglobules may be assessed by adding a targeting sequence to a dispersionof lipid globules (such as a mixture of phospholipid andtriacylglycerol) in an aqueous solvent, sonicating the mixture anddetermining by fractionation the degree of partition between aqueous andlipid phases. Typically fractionation of the mixture would involveincreasing the density of the solution with sorbitol or sodium bromideand ultracentrifuging the solution. The lipid complexes migrate to thetop of the centrifuge tube and this upper lipid layer is then examinedfor targeting sequence. This is performed in the presence or absence ofthe candidate substance whose inhibitory activity it is desired to test.Typically, a candidate substance is deemed to inhibit the lipid globuletargeting sequence if it causes a reduction of at least 50%, preferablyat least 60, 70, 80 or 90% in the amount of targeting sequenceassociated with the lipid phase in the absence of the candidatesubstance.

[0127] The candidate substance may be pre-incubated with the lipidglobule targeting sequence or with a lipid globule or added to thereaction mixture after pre-incubation of the lipid globule targetingsequence with a lipid globule.

[0128] Another type of in vitro assay for identifying substances whichdisrupt an interaction between the lipid globule targeting sequence anda lipid globule or a lipophilic surface involves the following:

[0129] Proteins incorporating the lipid globule targeting sequence, forexample a fragment of HCV core protein, and optionally ADRP, may betranslated in vitro from RNA transcripts encoding these polypeptides.Reactions would be supplemented with membranes originating from tissueculture cells (e.g. microsomal membranes) or a lipophilic surface whichhad been artificially created (e.g. a liposome) to which the in vitrotranslated proteins can bind. Binding may be assessed by purification ofthe lipid components of reactions (e.g. by centrifugation).Alternatively a chip format (for example BIAcore™—Biacore AB) whereinthe chip has a lipid surface may be used to assess binding.

[0130] The ability of candidate substances to disrupt association of thetargeting sequence and/or ADRP with lipid can be examined by adding toreactions candidate substances during synthesis of the proteins. Thus,the effects of substances on core/lipid globule targeting sequences andADRP association with lipid can be compared. Preferably, a suitableinhibitor of a lipid globule targeting sequence is capable of inhibitingbinding of viral lipid targeting sequences such as HCV core to lipidglobules without affecting binding of ADRP.

[0131] In vivo Assay Systems

[0132] In vivo assays for identifying compounds that disrupt aninteraction between the lipid globule targeting sequence and a lipidglobule typically involve administering a candidate substance to a cellwhich expresses a lipid globule targeting sequence, for example afragment of HCV core protein, and determining if the ability of thetargeting sequence to associate with intracellular lipid globules hasbeen affected, for example reduced or abolished. Thus the association ofthe targeting sequence with intracellular lipid globules is determinedin the absence of the candidate substance and in the presence of thecandidate substance and the results compared.

[0133] Association of the lipid globule targeting sequence withintracellular globules is typically determined by immunofluorescencemicroscopy using an antibody which recognizes the targeting sequence andan antibody or stain which recognizes intracellular lipid globules or asurface component of the globules. If the targeting sequences is able tobind to the lipid globules, then the targeting sequence willsubstantially co-localize with the lipid globules. A suitable procedureis described in the Examples. In addition, since we show that HCV coreprotein can displace ADRP from lipid globules, the co-localization ofendogenous ADRP with lipid globules may also be determined in thepresence and absence of the candidate substance.

[0134] A candidate substance is generally considered to be capable ofdisrupting the interaction between the lipid globule targeting sequenceand intracellular lipid globules if, as determined by immunofluorescencemicroscopy, less than 50% of the detectable targeting sequence proteinco-localizes with intracellular lipid globules, preferably less than60%, more preferably less than 70, 80 or 90%. Preferably, a suitableinhibitor of a lipid globule targeting sequence is capable of disruptingthe interaction of viral lipid targeting sequences such as HCV core withintracellular lipid globules/surface components without affecting theinteraction of ADRP with intracellular lipid globules/surfacecomponents.

[0135] It will be appreciated by the skilled person that othertechniques are available for determining localization of proteins andlipid within intact cells and that these techniques are also applicableto the assays of the present invention.

[0136] The candidate substance, i.e. the test compound, may beadministered to the cell in one or more ways. For example, it may beadded directly to the cell culture medium or injected into the cell.Alternatively, in the case of polypeptide candidate substances, the cellmay be transfected with a nucleic acid construct which directsexpression of the polypeptide in the cell. Preferably, the expression ofthe polypeptide is under the control of a regulatable promoter, forexample so that expression is induced shortly before it is desired toadminister the candidate substance.

[0137] Another suitable test may involve introducing a polynucleotideencoding a targeting sequence of the invention, optionally linked to aprotein of interest, into a milk-producing cell in culture anddetermining whether, the targeting sequence/protein of interest has beensecreted into the culture medium. This would be carried out in thepresence or absence of the candidate substance.

[0138] 2. Assays for Substances Capable of Modulating ADRP Expression inCells

[0139] i. Candidate Substances

[0140] Substances that modulate ADRP expression, preferably up-regulateADRP expression so that the levels of ADRP protein in a cell areincreased, may do so by one or more of several mechanisms. For example,a substance may increase levels of transcription from endogenous ADRPgenes, stabilizes ADRP mRNA levels and/or stabilizes ADRP protein.Transcription may be increased from endogenous ADRP genes by, forexample, the use of a transcriptional activator that activatestranscription from endogenous ADRP genes or a substance that modifiesthe effect of a transcriptional inhibitor of endogenous ADRP genes. Thustests for modulation of ADRP expression include determining the effectof a candidate substance on ADRP mRNA levels and/or ADRP protein levels.

[0141] The term “modulate” in the context of the ADRP expression means achange or alteration in the levels of ADRP mRNA and/or ADRP protein.

[0142] Suitable candidate substances include substances known tomodulate cellular transcription and/or lipid metabolism. Examples ofsubstances which may affect ADRP expression include non-steroidalanti-inflammatory drugs such as ibuprofen and indomethacin, and drugsknown to affect lipid metabolism such as fibrates andthiazolidinediones. Furthermore, combinatorial libraries, peptide andpeptide mimetics, in particular peptides from panels of peptidescomprising random sequences or sequences which have been variedconsistently to provide a maximally diverse panel of peptides, definedchemical entities such as organic and inorganic compounds,oligonucleotides, and natural product libraries may be screened foractivity as modulators of ADRP expression in assays such as thosedescribed below. The candidate substances may be used in an initialscreen in batches of, for example 10 substances per reaction, and thesubstances of those batches which show inhibition tested individually.In addition, candidate substances which show activity in in vitroscreens such as those described below can then be tested in in vivosystems, such as mammalian cells which will be exposed to the inhibitorand tested, for example, for susceptibility to viral infection.

[0143] ii. Assays

[0144] The assays of the invention may be in vitro assays or in vivoassays, for example using cell lines or an animal model.

[0145] In vitro Assay Systems

[0146] An in vitro assay system of the invention typically measures theeffect on transcription from a polynucleotide construct comprising anADRP promoter linked to a polynucleotide whose transcribed, andoptionally translated, product is capable of detection, for example thenaturally occurring ADRP coding sequence or a reporter gene such asluciferase. Techniques for detecting and quantitating transcriptionproducts are well known in the art and include, for examplehybridisation to labeled probes and direct quantitation of transcribedproducts by the use of labeled nucleotides which are incorporated duringtranscription. Translated products may also be detected using well knowntechniques such as SDS-PAGE and Western blotting, or in the case ofbiologically active products, suitable assays for detecting saidactivity (such as CAT assays or chemiluminescence assays).

[0147] A suitable in vitro assay may for example, be conducted usingextracts of mammalian cells, typically supplemented with buffers andnucleotide mixes. Reporter constructs comprising polynucleotidescontaining ADRP promoter constructs linked to a reporter gene may beadded to the mix, or endogenous genomic DNA used.

[0148] The effect of a candidate substance on ADRP expression may bedetermined by measuring levels of transcription from the ADRP promoterconstruct (endogenous or otherwise) in the presence and absence of thecandidate substance and comparing the results. Preferably, a controlpromoter construct should also be tested to ensure that any effect onADRP expression is specific to the ADRP promoter and is not simply theresult of general transcriptional inhibition. A candidate substance istypically considered to modulate ADRP expression if the levels oftranscriptional are altered by at least 30%, preferably at least 50, 60,70, 80 or 90%. Any affect on the control promoter should preferably beaccounted for when calculating changes in ADRP transcription.

[0149] In vivo Assay Systems

[0150] Modulation of ADRP expression can also conveniently be measuredin vivo, typically using mammalian cell lines. As with in vitro systems,both reporter constructs and endogenous ADRP genes may be used. In apreferred embodiment, the assay uses mammalian cells stably transfectedwith a polynucleotide comprising a reporter construct which contains anADRP promoter operably linked to a reporter gene, for examplechloramphenicol transferase (CAT) or luciferase. The cell alsopreferably comprises a stably transfected control promoter sequenceoperably linked to a second reporter gene which can be distinguishedfrom the first reporter gene. Typically, levels of transcription fromthe ADRP construct and the control construct are measured in the absenceof a candidate substance and then measured in the presence of acandidate substance. The effect of the candidate substance ontranscription from the ADRP reporter construct (or endogenous ADRP gene)can then be determined, taking into account any general effect ontranscription as indicated by the result obtained for the controlreporter construct.

[0151] In another embodiment, ADRP expression is measured by determiningthe amount of ADRP protein in cells before administration of thecandidate substance and after administration of the candidate substance.As described above, protein levels are typically measured by analysingcell extracts by SDS-PAGE and detecting the ADRP protein using Westernblotting. Alternatively, the cells used in the assay of the inventionmay comprise a reporter construct containing an ADRP promoter operablylinked to a nucleotide sequence encoding a detectable polypeptideproduct, such as CAT. In a particularly preferred embodiment, thedetectable product encodes an enzyme which can cleave a cellular orexogenously added substance causing a detectable change in theabsorption spectrum or emission spectrum of the cell or cell medium at aparticular wavelength. This will facilitate the large-scale screening ofcandidate substances in, for example a microtiter plate assay format.

[0152] Administration of candidate substances to mammalian cell linesand generation of host mammalian cell lines comprising reporterconstructs can be carried out as described above.

[0153] 3. Testing Candidate Substances for Anti-viral Activity

[0154] Candidate substances that are identified by the method of theinvention as disrupting an interaction between a lipid globule targetingsequence and a lipid globule, or modulating ADRP expression may betested for their ability to, for example, reduce susceptibility of cellsto viral infection. Such compounds could be used therapeutically toaffect viral infection, for example to prevent or treat viral infection.

[0155] Typically, an assay to determine the effect of a candidatesubstance identified by a method of the invention on the susceptibilityof cells to viral infection comprises:

[0156] (a) administering a virus, for example HCV, to a cell, in theabsence of the candidate substance;

[0157] (b) administering the virus to the cell in the presence of thecandidate substance; and

[0158] (c) determining if the candidate substance reduces or abolishesthe susceptibility of the cell to viral infection.

[0159] The candidate substance may be administered before, orconcomitant with, the virus to establish if infection is prevented.Alternatively, the candidate substance may be administered subsequent toviral infection to establish if viral infection can be treated using thecandidate substance. Administration of candidate substances to cells maybe performed as described above.

[0160] The assay is typically carried out using mammalian cell lines,but an animal model could be employed instead, such as chimpanzees inthe case of HCV. The virus is contacted with cells, typically cells inculture. The cells may be cells of a mammalian cell line, in particularmammalian cells susceptible to infection by the virus in the absence ofthe candidate substance, for example in the case of HCV, liver cells.

[0161] Techniques for assaying infectivity of viruses are well-known inthe art. As well as using plaque assays, levels of viral infection canbe determined by using recombinant viruses which comprise a reportergene, for example lacZ. The use of a histochemically detectable reportergene is especially preferred when experiments are performed withanimals. In the case of HCV, plaque assays cannot be used. A suitablemethod for determining the level of HCV production in an infected animalis to perform RT-PCR analysis of the amount of positive-strand nucleicacid material in cells or serum. In addition, the presence ofnegative-strand RNA in cells, as determined by RT-PCR, is taken to meanthat there is active viral replication.

[0162] In a preferred embodiment of the above-described assays, lipidglobule targeting sequence and derivatives thereof are used in anexperimental system to study normal cellular interactions. For example,derivatives of viral proteins such as HCV core protein or derivatives ofADRP, including deletion, insertion and substitution mutants, can beused to disrupt an interaction between ADRP and lipid globules. This canbe tested in vitro or in vivo using the assays described above. Theinteraction between ADRP and lipid globules can also be disrupted invivo by introducing a lipid globule targeting sequence or derivativesthereof, including deletion, insertion and substitution mutants, intocells in vivo, preferably mammalian cells, more preferably human cells.

[0163] Lipid globule targeting sequences and their derivatives can beintroduced into the cells using techniques described above, for exampletransfection of nucleic acid constructs encoding lipid globule targetingsequences, or using viral vectors. The effect of this disruption can bedetermined as described above. Any in vitro data obtained may be used toassist in the rational design of lipid globule targeting sequences foruse in the in vivo studies. In addition, the precise regions/amino acidresidues of lipid globule targeting sequences which bind to lipidglobules can determined by in vitro binding studies using lipid globuletargeting sequence derivatives. This will also assist in the rationaldesign of lipid globule targeting sequence derivatives for use in the invivo studies.

[0164] Thus viral lipid globule targeting sequences, which are readilydistinguished from cellular constituents, may be used as a tool toinvestigate intracellular lipid metabolism.

[0165] F. Therapeutic Uses

[0166] Substances capable of disrupting an interaction between a virallipid globule targeting sequence and an intracellular globule may beused to affect a viral infection in a human or animal, in particular totreat or prevent a viral infection. Such substances may have beenidentified by the assay methods of the invention or otherwise.

[0167] Substances that are capable of modulating ADRP expression mayalso be used to affect a viral infection in a human or animal, inparticular to treat or prevent a viral infection. Such substances mayhave been identified by the assay methods of the invention or otherwise.For example, the non-steroidal anti-inflammatory drugs ibuprofen andindomethacin have been shown to stimulate ADRP expression. Consequently,the present invention provides the use of ibuprofen and/or indomethacinin the manufacture of a medicament for use in affecting a viralinfection, preferably an HCV infection.

[0168] G. Compositions/Administration

[0169] Proteins of the invention and substances identified oridentifiable by the assay methods of the invention may preferably becombined with various components to produce compositions of theinvention. Preferably the compositions are combined with apharmaceutically acceptable carrier or diluent to produce apharmaceutical composition (which may be for human or animal use).Suitable carriers and diluents include isotonic saline solutions, forexample phosphate-buffered saline. The composition of the invention maybe administered by direct injection. The composition may be formulatedfor parenteral, intramuscular, intravenous, subcutaneous, intraocular ortransdermal administration. Typically, each protein may be administeredat a dose of from 0.01 to 30 mg/kg body weight, preferably from 0.1 to10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight.

[0170] Polynucleotides/vectors encoding polypeptide components for usein affecting viral infections may be administered directly as a nakednucleic acid construct, preferably further comprising flanking sequenceshomologous to the host cell genome. When the polynucleotides/vectors areadministered as a naked nucleic acid, the amount of nucleic acidadministered may typically be in the range of from 1 μg to 10 mg,preferably from 100 μg to 1 mg.

[0171] Uptake of naked nucleic acid constructs by mammalian cells isenhanced by several known transfection techniques for example thoseincluding the use of transfection agents. Example of these agentsinclude cationic agents (for example calcium phosphate and DEAE-dextran)and lipofectants (for example lipofectam™ and transfectam™). Typically,nucleic acid constructs are mixed with the transfection agent to producea composition.

[0172] Preferably the polynucleotide or vector of the invention iscombined with a pharmaceutically acceptable carrier or diluent toproduce a pharmaceutical composition. Suitable carriers and diluentsinclude isotonic saline solutions, for example phosphate-bufferedsaline. The composition may be formulated for parenteral, intramuscular,intravenous, subcutaneous, intraocular or transdermal administration.

[0173] The routes of administration and dosages described are intendedonly as a guide since a skilled practitioner will be able to determinereadily the optimum route of administration and dosage for anyparticular patient and condition.

[0174] The invention will be described with reference to the followingExamples which are intended to be illustrative only and not limiting.The Examples refer to the above-mentioned Figures.

FIG. 1: Analysis of the Core Proteins Made by the pSFV and pgHCVConstructs

[0175] A. Western blot analysis of extracts prepared from cells whichwere harvested 20 hours after electroporation. Aliquots of extractscontaining the same number of cell equivalents were analyzed withantibody JM122. The samples were from cells electroporated with RNA fromthe following constructs: lane 1, pSFV.1-195; lane 2, pSFV.1-173; lane3, pSFV.1-169; lane 4, pSFV.1-153; lane 5, pSFV.Δ155-161; lane 6,pSFV.Δ161-166; lane 7, no RNA.

[0176] Arrows denote the forms of core which have (labeled C) and havenot (labeled UC) been cleaved at the internal processing site.

[0177] B. In vitro translation of core proteins. Products of reactionswere electrophoresed on a 10% polyacrylamide gel and detected byautoradiography. The samples were from reactions containing thefollowing constructs: lane 1, pgHCV.1-195; lane 2, pgHCV.1-173; lane 3,pgHCV.1-153.

FIG. 2: Confocal Images of the Intracellular Localization of CoreProteins and Lipid Droplets

[0178] BHK C13 cells were harvested 20 hours after electroporation andfixed with 4% paraformaldehyde, 0.1% Triton X-100. Indirectimmunofluorescence was performed with antibody JM122 and an anti-mousesecondary antibody conjugated with FITC. Lipid droplets were stainedwith oil red O. Panels A, D, G, J, M, P, S and V show the distributionsof core protein. Panels B, E, H, K, N, Q, T and W show the locations oflipid droplets. Panels C, F, I, L, O, R, U and X are merged images ofcore protein and lipid droplets. Cells were electroporated with RNA fromthe following constructs: panels A, B and C, pSFV.1-195; panels D, E andF, pSFV.1-173; panels G, H and I, pSFV.1-169; panels J, K and L,pSFV.1-153; panels M, N and O, pSFV.Δ155-161; panels P, Q and R,pSFV.Δ161-166; panels S, T and U, pSFVA125-144; panels V, W and X,pSFV.1-124, 145-152.

FIG. 3: Effect of Expression of Core Proteins on the Ability to DetectADRP in BHKC13 Cells by Confocal Microscopy

[0179] Cells were harvested 20 hours after electroporation and fixedwith methanol. ADRP was detected with anti-adipophilin antibody and coreprotein with 308 antisera. Secondary antibodies were an anti-mouse IgGconjugated with FITC (for anti-adipophilin) and anti-rabbit IgGconjugated with Cy5 (for 308 antisera). Panels A, D, G, J, M and P areimages of ADRP localization. Panels B, E, H, K, N, and Q are images ofcore distribution. Panels C, F, I, L, O and R show the merged images ofcore and ADRP distributions. Cells were electroporated with RNA from thefollowing constructs: panels A, B and C, pSFV.1-195; panels D, E and F,pSFV.1-173; panels G, H and I, pSFV.1-169; panels J, K and L,pSFV.1-153; panels M, N and O, pSFV.Δ155-161; panels P, Q and R,pSFV.Δ161-166.

FIG. 4: Effect of Expression of Core Proteins on the Ability to DetectADRP in MCA RH7777 Cells by Confocal Microscopy

[0180] Cells were examined as described in the legend for FIG. 3.

FIG. 5: Effect of Expression of Core Protein on the Abundance of ADRP

[0181] BHK C13 cells were electroporated with RNA from pSFV.1-195 andpSFV.1-153 and extracts were prepared at the times indicated followingelectroporation. Aliquots of cell extracts were electrophoresed on 10%polyacrylamide gels and then the proteins were transferred tonitrocellulose membrane for Western blot analysis. The upper panels showmembranes probed with JM122 antibody while, in the lower panels,membranes were probed with anti-adipophilin antibody. Bandscorresponding to core proteins, expressed from pSFV.1-195 andpSFV.1-153, and ADRP are arrowed.

FIG. 6: Effect of Lipid Globule Targeting Sequences on Stability andCleavage of Core Protein

[0182] A. Western Blot analysis of extracts prepared from cells whichwere harvested 16 hours after electroporation following treatment withproteasomal inhibitor MG132. Aliquots of extracts containing the samenumber of cell equivalents were analyzed with antibody JM122. Thesamples were from cells electroporated with RNA from the followingconstructs: lanes 1 and 2, PSFV.1-195; lanes 3 and 4, pSFV.1-124,145-152; lanes 5 and 6, pSFV.Δ125-144; lanes 7 and 8, pSFV.1-153; lanes9 and 10, no RNA. Samples in lanes with odd numbers were prepared fromcells not treated with MG132. Samples in lanes with even numbers wereprepared from cells which had been incubated in the presence of MG132(final concentration 2.5 μM) from 4 hours after electroporation.

[0183] B. Western blot analysis of extracts prepared from cells whichwere harvested 16 hours after electroporation. Aliquots of extractscontaining the same number of cell equivalents were analyzed withantibody JM122. The samples were from cells electroporated with RNA fromthe following constructs: lane 1, pSFV.1-195; lane 2, pSFV.1-124,145-152; lane 3, pSFV.1-153; lane 4, pSFV.Δ125-144. The two forms ofcore protein produced by pSFV.Δ125-144 which have been either cleaved ornot cleaved (C and UC respectively) at the internal processing site arearrowed.

EXAMPLES

[0184] Cell Lines

[0185] Baby hamster kidney (BHK) C13 cells were maintained in Glasgowmodified Eagle's medium supplemented with 10% newborn calf serum, 100IU/ml penicillin/streptomycin and 5% tryptose phosphate broth. The rathepatoma cell line, MCA RH7777, was maintained in minimal essentialEagle's medium supplemented with 20% foetal bovine serum, 100 IU/mlpenicillin/streptomycin, 1 x non-essential amino acids and 2 mML-glutamine.

[0186] Immunological Reagents

[0187] Antibody JM122 was a mouse monoclonal antibody raised against apurified fusion protein, expressed in bacteria, which was composed ofthe N-terminal 118 amino acid residues of core protein encoded by HCVstrain Glasgow linked to a histidine tag. Antisera 308 was raised inrabbits against a branched peptide ([A/P]KPQRKTKRNT[I/N]RRPQDVKFPGG)₈K₇A(SEQ ID No. 6). The peptide consists of residues 5-27 of core proteinencoded by HCV strain Glasgow (SEQ ID No. 1). The two degenerate sitesat positions 1 and 12 were introduced to obtain antisera which would bereactive against core proteins from other isolates. The adipophilinantibody was obtained from Cymbus Biotechnology Ltd.

[0188] Secondary antibodies were obtained from Sigma with the exceptionof Cy5 conjugated goat anti-rabbit IgG which was obtained from Amersham.

[0189] Construction of Plasmids

[0190] Plasmids containing the coding region for the core protein of HCVstrain Glasgow were obtained by combining fragments from two constructscalled core.pTZ 18 and 5′-ΔNS2 (provided by M. McElwee and R. Elliott).Core.pTZ18 possesses nucleotide residues 7-615 of SEQ ID No. 1 of theHCV strain Glasgow genome and 5′-ΔNS2 contains residues 1-2895 fromreference. DNA fragments from these plasmids were combined in a vectorcalled pGEM1 to give a construct termed pgHCV.CE1E2. This plasmidcontains nucleotide residues 337-2895 from reference of the HCV strainGlasgow genome and therefore encodes the core, E1 and E2 proteins ofthis isolate.

[0191] For cloning purposes, the sequences immediately upstream ofresidue 337 (nucleotide 7 of SEQ ID No. 1) were modified to contain therecognition sequences for Bgl II and Kpn I restriction enzyme sites andimmediately downstream of residue 2895, an oligonucleotide was insertedwhich encodes a translational stop codon followed by the sequences for aBgl II restriction enzyme site. To create pgHCV.CElE2, a Bgl II DNAfragment containing the core, E1 and E2 sequences was inserted into theBam HI site of pGEM1; this plasmid was further modified by introducing aBgl II enzyme site at the Eco RI site in the pGEM backbone. Constructionof a derivative plasmid, pgHCV.1-195 was achieved by inserting anoligonucleotide (GCTGAGATCTA—SEQ ID No. 7) that had both a translationalstop codon and the sequences for a Bgl II enzyme site between a Fsp Ienzyme site at residue 925 (625 of SEQ ID No. 1) in the HCV genome and aHind III enzyme in the pGEM backbone. Thus, pgHCV.1-195 encodes theN-terminal 195 amino acids of HCV strain Glasgow. The nucleotide andpredicted amino acid sequence of this region of HCV strain Glasgow isshown in SEQ ID. No. 1. From pgHCV.1-195, the following series ofconstructs were made which had various regions of the HCV coding regionremoved (in 1 to 3 and 6, the numbers following pgHCV represent theamino acid residues of HCV strain Glasgow encoded by each construct):

[0192] 1. pgHCV.1-173 was constructed by inserting an oligonucleotideGTAACCTTCCTGGTTGCTCTTGAGATCTA (SEQ ID No. 8) between the Bst EII (atnucleotide residue 841 in the HCV strain Glasgow genome) and Hind IIIenzyme sites (located in the pGEM backbone) in pgHCV.1-195.

[0193] 2. pgHCV.1-169 was constructed by inserting an oligonucleotideGTAACCTTTGAGATCTA (SEQ ID No. 9) between the Bst EII (at nucleotideresidue 841 in the HCV strain Glasgow genome) and Hind III enzyme sites(located in the pGEM backbone) in pgHCV.1-195.

[0194] 3. pgHCV.1-153 was constructed by inserting an oligonucleotideCTGGCGCATTGAGATCTA (SEQ ID No. 10) between the Bst XI (at nucleotideresidue 492 of SEQ ID No. 1) and Hind III enzyme sites (located in thepGEM backbone) in pgHCV.1-195.

[0195] 4. pgHCV.Δ155-161 was constructed by inserting an oligonucleotideCTGGCCCATGGTGTTAACTATGCAACAG (SEQ ID No. 11) between the Bst XI and BstEII enzyme sites (at nucleotide residues 492 and 541 of SEQ ID No. 1respectively in the HCV strain Glasgow genome) in pgHCV.1-195. Thisconstruct lacks the nucleotide sequences encoding residues 155 to 161 ofthe core protein of HCV strain Glasgow.

[0196] 5. pgHCV.Δ161-166 was constructed by inserting anotheroligonucleotide CTGGCCCATGGCGTCCGGGTTCTGGAAGACG (SEQ ID No. 12) betweenthe Bst XI and Bst EII sites in pgHCV.1-195. This construct lacks thenucleotide sequences encoding residues 161 to 166 of the core protein ofHCV strain Glasgow from reference.

[0197] 6. pgHCV.1-124, 145-152 was constructed by inserting anoligonucleotide CGATAGAGGCGCTGCCAGGGCCCTGGCGTGAGATCTA (SEQ ID No. 13)between the Cla I (at nucleotide residue 410 from SEQ ID No. 1) and HindIII enzyme sites (located in the pGEM backbone) in pgHCV.1-195.

[0198] 7. pgHCV.Δ125-144 was constructed by inserting a 400 bp Kpn I/BstXI DNA fragment from pgHCV.1-124,145-152 (which contains residues 1-124and 145-152) into a 2970 bp Kpn I/Bst XI DNA fragment from pgHCV.1-195(which contains residues 153-195).

[0199] For expression in tissue culture cells, Bgl II DNA fragmentscarrying the relevant HCV sequences were prepared from the pgHCV plasmidseries and inserted into the Bam HI site of a Semliki Forest virusvector pSFV1. The resultant plasmids were termed the pSFV. series (e.g.pSFV.1-195).

[0200] In Vitro Translation

[0201] Proteins were translated in vitro using a coupledtranscription/translation kit supplied by Promega. Reactions used 1 μgof DNA as template and were carried out according to manufacturer'sinstructions.

[0202] In Vitro Transcription

[0203] Prior to electroporation, RNA was transcribed in vitro from theappropriate pSFV plasmid which had been linearized at a Spe I enzymesite. Typical reactions were carried out in a volume of 20 μl andcontained 40 mM Tris (pH 7.5), 6 mM MgCl₂, 2 mM spermidine, 10 mM NaCl,1 mM DTT, 1 mM ATP, 1 mM CTP, 1 mM UTP, 0.5 mM GTP, 1 mM m⁷G(5′)ppp(5′)Gcap analogue, 50 units Rnasin, 50 units SP6 RNA polymerase and 2 μglinearized DNA. Reactions were performed at 37° C. for 2 hours. Productsof the reaction were analyzed by agarose gel electrophoresis to examinethe quality and quantity of RNA synthesized prior to use inelectroporations.

[0204] Preparation of Cells Competent for Electroporation

[0205] Cells were washed and treated with trypsin for detachment fromtissue culture containers. Detached cells were suspended in 20 ml ofgrowth medium and centrifuged at 100 g for 5 min at room temperature.Cell pellets were suspended in 50 ml of PBSA and centrifuged aspreviously. Pellets were suspended in PBSA at a final concentration ofabout 2×10⁷ cells/ml.

[0206] Electroporation of Cells and Preparation of Cell Extracts

[0207] 0.8 ml of competent cells were mixed with in vitro transcribedRNA in an electroporation cuvette (0.4 cm gap) and pulsed twice ateither 1.2 kV, 25 μF (for BHK C13 cells) or 0.36 kV, 960 μF (for MCARH7777 cells). Between pulses, the cell/RNA suspension was gently mixed.Following electroporation, cells were diluted in growth medium andseeded onto either tissue culture dishes or coverslips in 24-well tissueculture plates and then incubated at 37° C.

[0208] To prepare extracts, electroporated cells were harvested byremoving the growth medium and washing the cell monolayers with PBS.Cells were scraped into PBS and pelleted by centrifugation at 100 g for5 mins at 4° C. The cell pellet was solubilized in sample bufferconsisting of 160 mM Tris (pH 6.7), 2% SDS, 700 mM β-mercaptoethanol,10% glycerol, 0.004% bromophenol blue.

[0209] Alternatively, sample buffer was added directly to cells whichhad been washed with PBS. Cells were solubilized at a concentration ofapproximately 4×10⁶ cell equivalents per ml sample buffer. Samples wereheated to 100° C. for 5 mins to fully denature proteins and nucleicacids.

[0210] SDS-PAGE and Western Blot Analysis

[0211] Samples were prepared for electrophoresis and proteins wereseparated on polyacrylamide gels cross-linked with 2.5% (wt/wt)N,N′-methylene bisacrylamide using standard techniques. Polypeptideswere detected either by autoradiography or by staining using Coomassiebrilliant blue.

[0212] For Western blot analysis, proteins were separated onpolyacrylamide gels and transferred to nitrocellulose membrane usingstandard techniques. The nitrocellulose membrane was blocked in 3%gelatin, 20 mM Tris (pH 7.5), 500 mM NaCl for at least 6 hours at 37° C.prior to incubation with the primary antibody. Incubations with theprimary antibody (diluted to {fraction (1/500)}for adipophilin antibodyand {fraction (1/1000)}for JM122) were performed in 1% gelatin, 20 mMTris (pH 7.5), 500 mM NaCl, 0.05% Tween 20 at either room temperature or37° C. for approximately 3-4 hours. Following extensive washing with 20mM Tris (pH 7.5), 500 mM NaCl, 0.05% Tween 20, the membrane wasincubated for 2 hours at room temperature with anti-mouse IgG conjugatedwith horse radish peroxidase in the same solution as for the primaryantibody and at a dilution of {fraction (1/1000)}. Bound antibody wasdetected by enhanced chemiluminescence.

[0213] Indirect Immunofluorescence and Staining of Lipids

[0214] Cells on 13 mm coverslips were fixed in either methanol at −20°C. or 4% paraformaldehyde, 0.1% Triton X-100 (prepared in PBS) at 4° C.for 30 mins. Following washing with PBS and blocking with PBS/CS (PBScontaining 1% newborn calf serum), cells were incubated with primaryantibody (diluted in PBS/CS at 1/200 for JM122 antibody, 1/1000 for 308antisera and 1/100 for adipophilin antibody) for 2 hours at roomtemperature. Cells were washed extensively with PBS/CS and thenincubated with conjugated secondary antibody (either anti-mouse oranti-rabbit IgG raised in goat) for 2 hours at room temperature. Cellswere washed extensively in solutions of PBS/CS followed by PBS andfinally H₂O before mounting on slides using Citifluor. Samples wereanalyzed using a Leiss LSM confocal microscope.

[0215] Following incubation with both antibodies and washing, lipiddroplets were stained in paraformaldehyde-fixed cells by briefly rinsingcoverslips in 60% propan-2-ol followed by incubation with 0.5 ml 60%propan-2-ol containing oil red O for 1.5-2 mins at room temperature.Coverslips were briefly rinsed with 60% propan-2-ol, washed with PBS andH₂O and mounted as described above. The oil red O staining solution wasprepared from a saturated stock of approximately 1% oil red O dissolvedin propan-2-ol. Before staining, the stock was diluted with H₂O and thenfiltered.

RESULTS Example 1 Expression of HCV Core Protein and Variants in TissueCulture Cells

[0216] Presently, there is no system available for propagating HCV intissue culture cells. Therefore, expression of HCV gene productsnecessitates the use of heterologous expression systems. For short-termexpression in mammalian cells, a variety of viral vectors have beenutilized including vaccinia virus, Sendai virus and adenovirus. Afurther alternative is the Semliki Forest virus (SFV) system in which invitro transcribed RNA, that encodes the SFV replication proteins as wellas a heterologous protein but not the SFV structural proteins, isintroduced into tissue culture cells. Introduction of nucleic acid intocells may be achieved by several routes but, in the examples given, themethod of choice is electroporation.

[0217] BHK cells were electroporated with RNA from the series of pSFVconstructs. 20 hours following electroporation, cells were harvested andextracts prepared. Samples were electrophoresed on a 10% polyacrylamidegel and, following electrophoresis, the proteins were transferred tonitrocellulose membrane for Western blot analysis.

[0218] Probing the membrane with the core-specific monoclonal antibodyJM122 revealed a major single species in each sample which correspondsto core protein. The apparent molecular weights of the proteins made bypSFV.1-195 and two truncated variants, pSFV.1-173 and pSFV.1-169, areapproximately 21 kDa and are almost identical (FIG. 1A, lanes 1-3).Cleavage between the core and E1 coding regions occurs between residues191 and 192. However, there is additional data which reveals that thecore protein is further processed by cleavage around residue 174(Moradpour et al., 1996) this cleavage site will be referred to as theinternal processing site. The precise residue at which this secondcleavage event occurs is not known. Hence, in agreement with FIG. 1A,lanes 1-3, it would be predicted that the three constructs named abovewould generate products of similar molecular weights.

[0219] Additional evidence for a cleavage event close to residues169-173 occurring within tissue culture cells is shown in FIG. 1B. Here,polypeptides translated in vitro from the pGEM versions of 3 corevariants reveal that the unprocessed species made from pgHCV.1-195 islarger than that from pgHCV.1-173 (compare lanes 1 and 2). As would bepredicted from the coding sequences for the third truncated form ofcore, the major species synthesized from pSFV.1-153 has a lower apparentmolecular weight than that from pSFV.1-195 (FIG. 1A, compare lanes 1 and4). The major species made by the internal deletion mutantspSFV.Δ155-161 and pSFV.Δ161-166 are intermediate in size between thoseproduced by pSFV.1-195 and pSFV.1-153 (FIG. 1A, lanes 5 and 6). Again,this agrees well with predictions based on the number of amino acidsremoved in these variants (7 in pSFV.Δ155-161 and 6 in pSFV.Δ161-166).It is also evident that there is a significant amount of materialproduced by the two internal deletion variants which has a highermolecular weight than the fully processed form of core. This presumablyrepresents reduced cleavage at the internal processing site which mayresult from the removal of certain residues in these mutants which arenecessary for fully efficient processing. To conclude, the core proteinsand its variants produced by the SFV constructs can be detected by acore-specific antibody and their apparent molecular weights are inagreement with predictions from the nucleotide sequences and previouslypublished data.

Example 2 Intracellular Distribution of HCV Core Protein

[0220] Previous studies have revealed that the HCV core protein canassociate with lipid droplets within the cytoplasm of cells (Barba, G.et al., 1997; Moradpour, D. et al., 1996). This conclusion was arrivedat by combining the techniques of immune electron microscopy with theability to stain lipid with osmium tetroxide. However, this methodsuffers from the disadvantages that it is time-consuming and osmiumtetroxide can stain other biological molecules (e.g. proteins) inaddition to lipid. Therefore, we developed a method for detectingproteins firstly by indirect immunofluorescence followed by staining oflipid droplets with the oil-soluble colorant oil red O. Combined withthe method of confocal microscopy, it is possible to visualize theintracellular localizations of core protein and lipid dropletsseparately and together. A typical example is shown in FIG. 2, panelsA-C. Here, BHK C13 cells have been electroporated with pSFV.1-195 RNAand, following incubation at 37° C. for 20 hours, the cells have beenexamined by both indirect immunofluorescence and staining with oil redO. In panel A, the core protein produced by pSFV.1-195 is seen to locateto vesicular structures in the cytoplasm. Panel B reveals thedistribution of lipid droplets in the same cell. By merging these data(panel C), it is evident that core protein is sited around the lipiddroplets. These data therefore agree with previously published resultsfor constructs expressing the full-length coding region of core.

Example 3 Association of HCV Core Protein with Intracellular LipidDroplets Requires Amino Acids 161 to 166 and 125 to 144

[0221] Results with the constructs which produce truncated forms of coreprotein indicate that proteins consisting of 173 and 169 amino acids ofthe core coding region also locate to droplets (FIG. 2, panels D-I). Bycontrast, expressing only the N-terminal 153 residues results in loss oflocalization to droplets and a diffuse cytoplasmic distribution isobserved (FIG. 2, panels J-L). Thus, residues of core protein betweenamino acids 154 and 169 are required for localization to droplets.Studies with the internal deletion mutants pSFV.Δ155-161 andpSFV.Δ161-166 further examined segments within this 16 amino acid regionwhich may be important for core protein localization. From the resultantdata, removal of residues between 155 and 161 did not affect lipiddroplet association whereas removal of residues between 161 and 166 gavea diffuse cytoplasmic pattern (FIG. 2, compare panels M-O with P-R).Hence, between residues 154 and 169, amino acids from 161 to 166 play anessential role in the ability of core protein to locate to lipiddroplets.

[0222] Further analysis of other internal deletion mutants (whichremoved residues 9-43, 4-75 and 80-118) showed that the core proteinsmade by these constructs continued to associate with lipid droplets(data not shown). Hence, these regions are dispensable for binding todroplets. However, a construct expressing a core variant in whichresidues 125 to 144 had been deleted failed to distribute to dropletsand gave a diffuse cytoplasmic fluorescence (FIG. 2, panels S, T and U).This mutant therefore identifies a second region in addition to thesegment between 161 and 166 which is necessary for association withdroplets. The data suggest that both sets of sequences are required fortargeting to lipid droplets. In agreement with these data, a corevariant which is truncated at residue 152 and lacks amino acids 125 to144 also fails to bind to droplets (FIG. 2, panels V, W and X).Additionally, this protein, in which both sets of targeting sequencesare deleted, is present in low amounts in electroporated cells as aconsequence of degradation.

Example 4 Effect of Localization of Core Protein on the Lipid DropletAssociated Protein ADRP

[0223] At present, there are few proteins identified in mammalian cellswhich are known to associate with lipid droplets. One protein which hasbeen recently identified is ADRP which is ubiquitously expressed in anumber of tissue culture cell lines; ADRP MRNA has also been detected ina range of tissue types in mice. To examine whether the localization ofcore to lipid droplets had any affect on ADRP, BHK C13 cells wereelectroporated with the series of pSFV constructs expressing coreprotein and its variants. An example of the data is shown in FIG. 3.Panels A to C show images of three cells following electroporation withpSFV.1-195, only one of which contains core protein (Panel B).Immunofluorescent results with the adipophilin antibody (panel A) revealthat ADRP is located on vesicular structures, consistent with itspreviously assigned association with lipid droplets. The protein isreadily detected in the cells which do not express core protein,however, it is considerably reduced in abundance in the core-expressingcell. Observations from this and a series of other experimentsconsistently revealed that cells expressing core protein from pSFV.1-195either lacked or contained barely detectable amounts of ADRP.Nonetheless, some cells in which both core protein and ADRP were presentalso were found; in general, such cells gave reduced fluorescence forthe core protein. Hence, it was concluded that the loss of ADRP wasrelated to the levels of expression of core in individual cells. Resultswith the variants of core which continue to locate to lipid dropletsgave identical data (see panels D-I and M-O). Thus, the majority ofcells expressing core protein from constructs pSFV.1-173, pSFV.1-169 andpSFV.Δ155-161 contained quantities of ADRP which were barely detectable.By contrast, ADRP continued to be readily found in cells producing coreproteins from pSFV.1-153 and pSFV.Δ161-166, the variants which do notassociate with lipid droplets (see panels J-L and P-R). Thus,association of core protein with lipid droplets correlates with a lossof ability to detect ADRP by immunofluorescence. Any cell typespecificity for this affect was tested by performing identicalexperiments in the rat hepatoma cell line, MCA RH7777. In these cells,core protein and its variants gave identical results for their abilityto associate with lipid droplets and this again correlated with thelevels of ADRP detected in core-expressing cells (FIG. 4). Thus theeffect of core protein on ADRP is not cell-type specific.

Example 5 The Ability of Core Protein to Associate with Lipid DropletsInduces a Loss of ADRP

[0224] The immunofluorescence data revealed that the association of coreprotein and its variants with lipid droplets led to an inability todetect ADRP. It was possible that this was due to masking of ADRP bycore. To examine directly the effect of core protein on the levels ofADRP, Western blot analysis was performed on cell extracts prepared atvarious times following electroporation with either pSFV.1-195 orpSFV.1-153 RNA. In parallel, immunofluorescence analysis was alsoperformed on these cells and this revealed that expression of the coreprotein produced by the two RNAs was apparent in greater than 90% ofcells. Analysis with antibody JM122 indicated that core protein could bedetected from both constructs at 10 hours following electroporation andpeaked by about 20 hours (FIG. 5). The abundance of core proteinproduced by the two constructs was very similar by this time-point. Fromanalysis of these samples with the ADRP-specific antibody, it isapparent that there is no change in the abundance of ADRP followingelectroporation with the pSFV. 1-153 RNA. A third set of cells in thisexperiment which was electroporated with SFV RNA which expresses the HCVE1 and E2 proteins also showed no reduction in ADRP levels with time. Bycontrast, there is a rapid reduction in ADRP levels to barely detectablequantities which mirrors the rise in core protein made from pSFV.1-195.

[0225] From staining of polyacrylamide gels with Coomassie brilliantblue, there were approximately equivalent amounts of protein in allsamples. In addition, probing the membranes with another antibody for aendoplasmic reticulum-specific protein, calnexin, indicated that bothpSFV.1-195 and pSFV.1-153 samples had similar quantities of this proteinat the various times following electroporation. This affect of coreexpression on ADRP was consistently found in other experiments. Thus,the association of core protein with lipid droplets directly correlateswith a specific reduction in the abundance of this protein in cells.

Example 6 Removal of Lipid Globule Targeting Sequences Reduces ProteinStability and Impairs Cleavage at the Internal Processing Site

[0226] Immunofluorescence data with pSFV.1-124,145-152 and pSFV.Δ125-144showed that the proteins made by these constructs gave diffusefluorescence. In two separate experiments, Western blot analysis ofextracts prepared from cells electroporated with RNA from theseconstructs revealed significantly reduced amounts of protein made bypSFV.1-124,145-152 as compared to that made by pSFV.1-195, pSFV.1-153and pSFV.Δ125-144 (FIG. 6A, compare lane 3 with lanes 1, 5 and 7 andFIG. 6B, compare lane 2 with lanes 1, 3 and 4).

[0227] The level of reduction was approximately 5-fold. This low levelof detectable protein was not the result of reduced amounts of RNAsynthesized by in vitro reactions (data not shown). Addition of theproteasome inhibitor MG132 to a parallel culture of cells electroporatedwith pSFV.1-124, 145-152 gave rise to a significant (4-fold) increase inthe amount of protein detected by Western blot analysis (FIG. 6A,compare lane 4 with lane 3). Therefore, the reduction in protein levelsfrom pSFV.1-124, 145-152 RNA is apparently not due to inefficienttranslation. These data suggest that removal of both sets of sequenceswhich are required for targeting to lipid droplets induces rapiddegradation of the protein, analogous to that observed for ADRP in thepresence of full-length core protein.

[0228] It was observed also that two protein products derived frompSFV.Δ125-144 were detected by antibody JM122. From their mobilities, itwas assumed that the upper band represented protein which had not beencleaved at the internal processing site (labeled UC in FIG. 6B, lane 4)while the lower band was processed (labeled C in FIG. 6B). Examinationof the relative intensities of these species indicates that theuncleaved product is more abundant (approximately 4-5 fold) than thecleaved material. This provides evidence that the sequences between 125and 144, which are required for lipid droplet association also influencethe efficiency of cleavage at the internal processing site. Cleavage atthis site is mediated by a cellular signalase which may be membranebound. Hence, disruption of the targeting function of these residues islikely to inhibit cleavage at the internal processing site and, fromevidence of the maturation of related pestiviruses and flaviviruses,such an effect will reduce virus maturation and growth.

Example 7 In Vitro Binding Assay to Test Effects of Candidate SubstancesSynthesis of Lipid Targeting Sequences and ADRP Using in VitroTranscription/Translation

[0229] Constructs suitable for in vitro synthesis of mRNA are producedby placing nucleotide sequences encoding core protein (e.g. pgHCV.1-195)or any other protein with core lipid globule targeting sequences (e.g.pgHCV.Δ43-119) and ADRP are placed under the control of a suitablebacterial or bacteriophage RNA polymerase promoter e.g. SP6 or T7. RNAtranscripts are prepared from each of these constructs using standardmethods and the yields of RNA determined.

[0230] To produce in vitro translated protein, the in vitro synthesizedRNA is added to an extract capable of synthesizing polypeptides e.g. areticulocyte lysate prepared from rabbits or an extract from wheat germ;proteins may be radiolabeled by including an isotopically labeled aminoacid such as ³⁵S-methionine.

[0231] Assessing Binding of Proteins to Lipid

[0232] Membranous material prepared from animal cells (e.g. microsomalmembranes) or synthetically prepared mixtures of triacylglycerol orcholesterol and phospholipid are mixed with the translated proteins andincubated. Lipid fractions are then recovered by centrifugation. Theincorporation of proteins into lipid fractions may be determined eitherdirectly by SDS-PAGE of radiolabeled proteins, or indirectly by usingantibodies which are functional in Western blot and/or ELISA procedures.

[0233] Combinations of RNA from core protein and ADRP may be mixed inthe same reaction in various proportions to test the relative affinityof each protein for lipid. An example of a combination would be 9 partspgHCV.1-195 RNA to 1 part ADRP RNA.

[0234] Candidate substances at a range of concentrations, such as from 1μM to 100 mM, are added to reactions and the effect of these substanceson protein binding to lipid analyzed using the methods indicated above.A suitable candidate substance is typically a substance that enhances ordoes not significantly impair lipid association of ADRP but whichreduces or abolishes binding of core to lipid.

Example 8 In Vivo Binding Assay to Test Effects of Candidate Substances

[0235] BHK C13 cells are electroporated with SFV encoding HCV coreprotein (for example SFV.1-195) as described above. At various timepoints after electroporation, candidate substances are added to cells atconcentrations which are not cytotoxic (as determined with mockelectroporated cells, for example). Following incubation at 37° C.,cells are harvested and cell extracts prepared. Extracts are analyzed byWestern blot analysis (antibody JM122) to examine the abundance of coreprotein and determine the efficiency of cleavage at the internalprocessing site. The relative abundance of core protein in treated ascompared to untreated cells could also be quantitated by ELISA.

[0236] In addition, cells are fixed and examined by a combination ofindirect immunofluorescence (for the core and ADRP proteins) and oil redO staining (for lipid droplets) as described above. This can be used todetermine whether the intracellular distribution and abundance of coreand ADRP has been altered by the presence of the candidate substances.Western blot analysis may also be used to confirm whether the candidatesubstance had any affect on ADRP levels.

[0237] A suitable candidate substance is typically a substance thatprevents or reduces core association with droplets, causes reducedlevels of core protein and/or impaired cleavage at the internalprocessing site. By contrast, the candidate substance should preferablynot affect the intracellular distribution of ADRP and its abundance istypically either similar to or higher than in cells that do not expressthe core protein.

Example 9 Assessing Anti-viral Affects of Candidate Substances inTransgenic Animals Expressing Core or in Chimpanzees

[0238] In transgenic animals expressing liver-specific core protein oranimals (chimpanzees) infected with HCV, test substances are added atconcentrations which were not toxic to the host.

[0239] Transgenic Animals Expressing Core Protein in a Liver-specificManner

[0240] Transgenic mice which give liver-specific expression of coreprotein are known in the art. Expression of core protein is associatedwith the development of steatosis and hepatocellular carcinoma in twolines of such animals (Moriya, K. et al., 1998 and Moriya, K. et al.,1997); both pathologies are associated with HCV infection. Similartransgenic mice may be produced with similar phenotypes and used toexamine the effect of substances which prevent core association withlipid droplets on these pathological changes.

[0241] The effect of candidate substances on core protein localizationand levels may be determined in transgenic animals using cells obtainedby liver biopsies and tested using the techniques described in Example8. Alternatively, or in addition, the effect of a candidate substance ofthe development of steatosis and hepatocellular carcinoma in theseanimals may be determined. The efficacy of candidate substances may bemeasured by a reduction in the pathological changes which occur e.g.reduced hepatosteatosis and significant delays or prevention of theonset of carcinoma.

[0242] Chimpanzees Infected with HCV

[0243] The effect of a substance on HCV replication in infectedchimpanzees is assessed by RT-PCR analysis of sera taken at regularintervals from the animal. Biopsy material from the liver may also betested for the presence of negative-strand HCV RNA by RT-PCR usingstandard techniques (see Conry-Cantilena, 1997 for review and referencescontained therein). Efficacy of the candidate substance is assessed bythe reduction in the levels of HCV RNA as measured in either or bothassays. A reduction in viral titer by a factor of at least 2 to 3 logsis indicative of an anti-viral effect.

Example 10 Identification of a Domain Required to Direct Core Protein ofHCV and GB Virus B to Lipid Droplets

[0244] In this example we have identified a conserved domain that ispresent in equivalent structural proteins encoded by HCV and GBV-B anddirects these proteins to lipid storage compartments.

[0245] GBV-B is a virus called GB virus-B. Information on GB virus-B hasbeen presented by Beames et al (2000, Journal of Virology vol 74 No. 24,pp 11764-11772) and Lanford et al (2001, Journal of Virology vol 75 No.17, pp 8074-8081).

[0246] GBV-B was isolated from tamarins but the natural host of thevirus is not known. The GBV-B core protein is described in the examplebelow. The nucleic acid sequence of GBV-B is shown in Genbank AccessionNo. NC001655. GBV-B is the closest known related virus to HCV in termsof sequence identity (29). GBV-B core protein is an example of ahomologue of HCV core protein. An alignment of HCV and GVB-B amino acidsequence is shown in FIG. 7. Furthermore, GVB-B is an example of a lipidglobule targeting sequence.

[0247] Lipid droplets are intracellular storage organelles that arefound in all eukaryotic organisms and some prokaryotes (reviewed in1-3). They consist of a core of neutral lipid, comprising mainlytriacylglycerols and/or cholesterol esters, surrounded by a monolayer ofphospholipids. Bounding the phospholipid layer is a proteinaceous coat.In mammalian cells, the principal lipid droplet binding proteins thathave been identified are adipophilin (also called adipocytedifferentiation-related protein, ADRP; 7, 8) and a related family ofproteins termed the perilipins (9-11). Adipophilin is present in a widerange of cell types and in increased quantities in certain diseaseswhere intracellular lipid accumulation is evident (12-14). By contrast,expression of the perilipins is restricted to adipocytes andsteroidogenic cells (15, 16).

[0248] In addition to adipophilin and the perilipins, the core proteinencoded by hepatitis C virus (HCV) also associates with lipid dropletsin mammalian cells (17-19). HCV is the sole member of the hepacivirusgenus that is incorporated into the Flaviviridae family along with twoother genera, the flavi- and pestiviruses. All of the Flaviviridae arepositive-sense, single stranded RNA viruses that have similar genomearrangements and share sequence similarities. HCV core is a structuralcomponent of the virus particle and, by analogy with flavi- andpestiviruses, it is likely to be the sole component of the capsid (20,21). The protein is generated from a polyprotein encoded by the viralgenome by cleavage at the endoplasmic reticulum (ER; 22-26). Expressionof core can lead to the genesis of lipid droplets in tissue culturecells (18) and the development of steatosis in transgenic mice (27).Moreover, interaction between core and apolipoprotein AII, a componentof lipid droplets, has been demonstrated (28). These data indicate that,not only does core associate with lipid droplets, but it also may havethe capacity to influence metabolic events within the cell involving thestorage of lipid.

[0249] To understand the significance of the interaction between HCVcore and lipid droplets, a region within the viral protein that isessential for its association with these storage organelles had beenidentified (19). In this example, studies have been carried out todetermine whether there is any similarity between the sequences withinthis region and those of GBV-B, which has significant sequence identityto HCV although, as yet, it remains unclassified among the genera of theFlaviviridae (29). GBV-B shares a tropism for liver hepatocytes with HCVand is infectious in tamarins (30, 31). Thus, it has been suggested thatGBV-B infection of tamarins may be a surrogate model system for HCVinfection of humans (31). However, few comparative studies betweenequivalent proteins encoded by the two viruses have been conducted.

[0250] Construction of Plasmids

[0251] Construction of plasmids pSFV/1-195 and pSFV/1-169 has beendescribed previously (19). The codons for the proline residues in theseconstructs were mutated to encode alanine by insertion of anoligonucleotide (HCV1 in Table 1). This oligonucleotide was insertedbetween BspHI and BstXI sites in construct pgHCV/135-144 (19) to giveplasmid pgHCV/1-195

(P

A); the BspHI site is not a natural site in the sequence for HCV strainGlasgow and was introduced during the construction of pgHCV/135-144. Togenerate pSFV/1-195

(P

A) and pSFV/1-169

(P

A), a 502bp fragment from pgHCV/1-195

(P

A), produced by cleavage by BglII and BstEII, was inserted intopSFV/1-195 and pSFV/1-169 digested with the same enzymes. A taggedversion of HCV core was generated by firstly converting the nucleotidesequence in pg/1-195 that encodes amino acids 116 and 117 from TCG CGCto TCT AGA; this introduced a novel XbaI site into the HCV core-codingregion without changing the encoded amino acids. An oligonucleotide(HCV2 in Table 1) that encoded the epitope tag (32) was introduced intothe XbaI site to give plasmid pg/1-195tag. The tagged version of corewas transferred as a BglII fragment from pg/1-195tag into Semliki Forestvirus (SFV) expression vector pSFV1 (33) to give constructpSFV/1-195tag. TABLE 1 Oligonucleotides used to generate constructs.Nucleotide Name Oligonucleotide Sequence Position^(a) HCVCATGGGGTACATAGCGCTCGTCGGCGCCGCCT 1 TAAGAGGCGCTGCGAGGGCC (SEQ ID No. 14)HCV CTAGAGAGCGCAAGACGCCCCGCGTCACCGG 2 CGGCG (SEQ ID No. 15) GBVGGAGATCTCGTAGACCGTAGCACATG 428-448 B1 (SEQ ID No. 16) GBVGGGGATCCCTAGTGGACACCGAACCAACCAG 842-868 B2 TAGCCCA (SEQ ID No. 17) GBVGGGGATCCTCAGATCACACAACCAGGCTCGT 1003-1029 B3 GTAGG (SEQ ID No. 18) GBVGGGTACTCTAGAGTGATAGGCCTGGTC 1618-1639 B4 (SEQ ID No. 19) GBVCTAGAGAGCGCAAGACGCCGCGGGTCACCGG B5 TGGCTCTCGCAATCTTGG (SEQ ID No. 20)

[0252] To express portions of the GBV-B polyprotein, relevant regionswere amplified by PCR from a construct pGBB (30). pGBB contains theconsensus sequence for an infectious molecular clone of GBV-B (30).Primers for PCR amplification were derived from the viral sequences inpGBB and were used in the following pairs to produce DNA fragments thatencoded N-terminal regions of the GBV-B polyprotein: residues 1-141,GBV-B primers 1 and 2; residues 1-194, GBV-B primers 1 and 3; residues1-398, GBV-B primers 1 and 4. Amplified fragments were introducedinitially into plasmid pGEM1 and thereafter into pSFV1 by standardcloning techniques. To permit detection of GBV-B core, an epitope tag(32) was introduced into the coding region immediately following aminoacid residue 85 (FIG. 7). This was accomplished by firstly introducing anovel XbaI site at nucleotide residue 695 by converting the sequencefrom TCT CGC to TCT AGA; this did not alter the encoded amino acidsequence. An oligonucleotide encoding the epitope tag (GBV-B5 inTable 1) was inserted between this XbaI site and a TfiI site (position708 in the native GBV-B nucleotide sequence). The final SFV constructsthat contained tagged versions of GBV-B core were termed pSFV/GB1-141,pSFV/GB1-194 and pSFV/GB1-398.

[0253] Maintenance of Tissue Culture Cells and Treatment with MG132

[0254] Baby hamster kidney (BHK) C13 cells were grown and maintained inGlasgow minimal Eagle's Medium supplemented with 10% new-born calf serum(CS), 4% tryptose phosphate broth, and 100 IU/ml penicillin/streptomycin(ETC10). To treat BHK cells with MG132 (supplied by Boston Biochem),cells were incubated for 5h after electroporation at 37° C. and themedia was replaced with fresh media containing the protease inhibitor ata final concentration of 2.5 μg/ml. Incubation was continued at 37° C.for a further 12 h before the cells were either harvested for Westernblot analysis or fixed for indirect immunofluorescence studies.

[0255] Immunological Reagents

[0256] The monoclonal antibodies (MAb) used to detect HCV core protein(MAb JM122) and the epitope tag (MAb 9220) have been describedpreviously (19 and 32 respectively).

[0257] In Vitro Transcription and Electroporation of SFV RNA into Cells

[0258] RNA was transcribed in vitro from recombinant pSFV constructslinearized with SpeI. BHK cells were electroporated with in vitrotranscribed RNA as described previously (19, 36). Cells were incubatedat 37° C. for 15h and then harvested.

[0259] Preparation of Cell Extracts, Polyacrylamide Gel Electrophoresisand Western Blot Analysis

[0260] Extracts were prepared and polyacrylamide gel electrophoresisperformed as described previously (19, 36).

[0261] For Western blot analysis, proteins separated on polyacrylamidegels were transferred to nitrocellulose membrane. After blocking with 3%gelatin, 4 mM Tris-HCl, pH 7.4, 100 mM NaCl, membranes were incubatedwith antibodies (diluted to {fraction (1/500)}) in 1% gelatin, 4 mMTris-HCl, pH 7.4, 100 mM NaCl, 0.05% Tween 20. After washing, boundantibody was detected using a horseradish peroxidase-conjugatedsecondary antibody followed by enhanced chemiluminescence (Amersham,UK).

[0262] Indirect Immunofluorescence and Staining of Lipids

[0263] Cells on 13 mm coverslips were fixed for 30 min in 4%paraformaldehyde, 0.1% Triton X-100 (prepared in phosphate-bufferedsaline) at 4° C. Following washing with phosphate-buffered saline (PBS)and blocking with PBS/CS (PBS containing 1% newborn calf serum), cellswere incubated with primary antibody (diluted in PBS/CS at {fraction(1/200)}for JM122 and 9220 antibodies) for 2h at room temperature. Cellswere washed extensively with PBS/CS and then incubated with conjugatedsecondary antibody (either anti-mouse or anti-rabbit IgG raised in goat)for 2 h at room temperature. Cells were washed extensively in solutionsof PBS/CS followed by PBS. Staining of lipid droplets by oil red O wasperformed as described previously (19). Cells were rinsed finally withH₂O before mounting on slides using Citifluor (Citifluor Ltd., UK).Samples were analyzed using a Zeiss LSM confocal microscope.

[0264] Computing

[0265] Sequences were aligned using the CLUSTAL W alignment program (37)and hydropathicity plots generated by ProtScale (38).

RESULTS

[0266] A Domain in the HCV Core Protein that is Absent in Flavi- andPestiviruses but Present in GBV-B

[0267] Previously, it was proposed that the HCV core protein consistedof 3 domains and that the second of these domains was absent in relatedpesti- and flaviviruses (19, 39). Although there is a lack of sequenceidentity between viral sequences in the Flaviviridae, each of the capsidproteins in members of this virus family have a high content of basicamino acids (40). Therefore, the comparisons described here were basedon the proportion of positively charged residues in predicted codingregions accompanied by hydropathicity plot studies. It was found thatthe mature capsid (C) protein of yellow fever virus (YF), a flavivirus,had a high proportion of basic residues (27%). In contrast, the signalpeptide sequence that is removed from C protein upon maturation did notcontain any basic amino acids. Up to amino acid 117 of the HCV coreprotein, 23% of residues were basic and this dropped to 7% betweenresidues 118-173. Thus, the N-terminal 117 amino acids of HCV core havea similar character to those in the YF C protein but there are nosequences corresponding to the region between 118 and 173 of the HCVpolypeptide. Analysis of the hydropathicity of the HCV core protein alsorevealed a highly hydrophilic region containing a similar segment ofbasic residues (RRRSR) between residues 113-117 (FIG. 7). The regionbetween residues 118 and 173 is referred to as domain 2 (19, 39).

[0268] A closely related virus to HCV is GBV-B, a virus that wasisolated from tamarins but whose natural host is not known. To date, theproteolytic events to generate the mature proteins of GBV-B have beenassumed from comparison with HCV polypeptide processing (29). Comparingthe putative GBV-B core sequence with that of HCV did not identify anystretches of similarity until residue 75 of GBV-B (FIG. 7). According tothe sequence alignment, domain 1 for GBV-B core ended at residue 85(corresponding to residue 117 in HCV core; FIG. 7) and thus, for thisdomain, the GBV-B sequence was shorter than that for HCV. The overallsequence identity (sequence homology) between these domains in HCV andGBV-B was about 22%. From residue 86 and up to residue 140 of GBV-B,sequence identity between the two viral sequences increased to 41% andapart from one additional residue in GBV-B, the sequences were colinear(FIG. 7). In HCV core, this segment is composed principally of domain 2and indicated that sequences corresponding to this region are present inGBV-B. Sequence identity between the residues 140-156 for GBV-B and174-191 for HCV (corresponding to the signal peptide sequences) wasslightly lower at 38% and reduced further in the equivalent El sequences(approx. 25%). Distribution of basic residues in the putative GBV-B coreprotein revealed a high lysine/arginine content (21%) in the N-terminalregion up to amino acid 85, a reduced percentage beyond this point (3.6%between amino acids 86-136) and no positively charged amino acids in thesignal peptide sequence. This pattern of distribution corresponded tothat present in HCV core. From these data, it was concluded that theputative GBV-B core protein shares the same domain arrangement as itscounterpart in HCV.

[0269] Intracellular Localization of the GBV-B Core Protein

[0270] Previous studies on the intracellular localization of HCV coreshowed that the protein was directed to lipid droplets and the primarysequence determinants for this localization were present in domain 2(19). Since the region of highest homology between the core proteins ofGBV-B and HCV encompassed this domain, we expressed a N-terminal regionof the GBV-B polyprotein using pSFV/GB 1-398 in which the coding regionextended beyond the predicted C-terminus of El to analyze theintracellular localization of GBV-B core. As immunological reagentsagainst GBV-B proteins were not available, we placed a short epitope tag(32) at residue 85 to detect the protein. Placing this tag into thecorresponding region of the HCV core protein did not affect itsintracellular localization and the protein was present on lipiddroplets. Indirect immunofluorescence of cells electroporated with RNAfrom pSFV/GB1-398 using an antibody, 9220, that recognizes the epitopetag revealed staining around lipid droplets stained with oil red O.Western blot analysis of cell extracts indicated that the size of coreprotein detected was approximately 17 kDa. This is consistent with aproduct of about 155 amino acids (comprising 143 amino acids of GBV-Band 12 amino acids of the epitope tag).

[0271] To further verify the intracellular localization of GBV-B coreand the likely events involved in its maturation, two other constructscontaining GBV-B sequences were examined. Firstly, a constructpSFV/GB1-141 that would correspond approximately in size to the productdetected with pSFV/GB1-398. Analysis of cells expressing the taggedproduct produced by pSFV/GB1-141 again revealed localization of theprotein around lipid droplets. The size of the protein made bypSFV/GB1-141 was only slightly smaller than that made by pSFV/GB1-398.Another construct that expressed the N-terminal 194 residues of GBV-Bgave a major product that was identical in size to that produced bypSFV/GB1-398; a second minor band of about 20 kDa represented uncleavedprotein. It was concluded that, in common with HCV core, the equivalentGBV-B protein is directed to lipid droplets in tissue culture cells andcleavage of the GBV-B polyprotein to produce the mature form of core isdirected by cellular peptidases. Our studies on HCV core revealed thatdomain 2 contained the sequences essential for lipid dropletassociation. Based on our sequence comparisons, this domain is presentalso in the equivalent GBV-B protein. Hence, we propose that theessential sequences for association with lipid droplets reside withinthe corresponding region of GBV-B core.

[0272] Discussion

[0273] From previous analysis and data presented in this example, it wasproposed that the HCV core protein consisted of three domains (19, 39).Domain 1 corresponded to the mature core protein of flaviviruses whileno sequences equivalent to domain 2 were present in either pesti- orflaviviruses. Domain 3 contained the signal peptide sequence thatdirects the HCV E1 glycoprotein to the ER lumen. Here, the sequencecomparisons were extended to include GBV-B, a virus that is the closestknown related virus to HCV in terms of sequence identity (29). Fromcomparisons of the two viral sequences, domain 2 in HCV core was foundalso in the corresponding GBV-B protein. Sequence identity between thepredicted amino acid sequences of HCV and GBV-B in the region containingdomain 2 was higher (approx. 41%) than that in the N- and C-terminalflanking regions. This degree of similarity would imply that thesedomains in HCV and GBV-B might perform similar functions. Extensivemutational analysis and immunolocalization studies of the HCV coreprotein showed that domain 2 contained the key elements for directingthe protein to lipid droplets (19). The data presented in this exampleindicated that the core protein of GBV-B also could associate with theselipid storage structures. Thus, we conclude that a function of thesedomains is to direct the core protein of the two viruses to lipidstorage organelles.

[0274] From the above evidence, it was concluded that the HCV and GBV-Bcore proteins have similar domain configurations. Based on our data (19and this example), domain 2 in both proteins represented sequences thatdirected them to lipid droplets. Comparison with mammalian proteins thatassociate with these structures did not identify any region in suchproteins with features similar to those in domain 2.

[0275] The HCV and GBV-B core proteins each contained two closely spacedproline residues within domain 2. Substitution of these prolines in HCVcore abolished lipid droplet association. We have shown previously thatremoval of short sequence elements of domain 2 from HCV core preventedlipid droplet association (19). Based on these findings, we suggest thatthe HCV and GBV-B core may be members of a family of proteins that sharesimilar sequence characteristics for targeting to lipid droplets.

[0276] To summarize this example, in mammalian tissue culture cells, thecore protein of hepatitis C virus (HCV) is located at the surface oflipid droplets, which are cytoplasmic structures that store lipid. Thecritical amino acid sequences necessary for this localization are in aregion of core protein that is absent in flavi- and pestiviruses, whichare related to HCV. From sequence comparisons, this region in HCV corewas present in the corresponding protein of GB virus-B (GBV-B), anothervirus whose genomic sequence has significant similarity (homology asdefined herein) to HCV. Expression of the putative GBV-B core proteinrevealed that it also was directed to lipid droplets. Extending thecomparisons to mammalian cellular proteins, there were no amino acidsimilarities with the domains for lipid droplet association in HCV core.

[0277] All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

General References

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[0281] Clayerie & States (1993) Computers and Chemistry 17:191-201

[0282] Conry-Cantilena, 1997, Tibtech 15: 71-76

[0283] Devereux et al, 1984, Nucleic Acids Research 12: 387

[0284] Engelman et al., 1986, Ann Rev, Biophys. Chem 15: 343

[0285] Heid et al., 1998, Cell Tiss Res 294: 309-321

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[0288] Moriya, K. et al., 1997, J. Gen. Virol. 78; 1527-1531

[0289] Patton, S. and Huston, G. E., 1986, Lipids 21; 170-174

[0290] Wootton & Federhen, 1993, Computers and Chemistry 17:149-163

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1 20 1 630 DNA Hepatitis C Virus CDS (43)...(630) 1 ggtgcttgcgagtgccccgg gaggtctcgt agaccgtgca cc atg agc acg aat 54 Met Ser Thr Asn 1cct aaa cct caa aga aaa acc aaa cgt aac acc aac cgt cgc cca cag 102 ProLys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Pro Gln 5 10 15 20gac gtt aag ttc ccg ggt ggc ggt cag atc gtt ggt gga gtt tac ttg 150 AspVal Lys Phe Pro Gly Gly Gly Gln Ile Val Gly Gly Val Tyr Leu 25 30 35 ttgccg cgc agg ggc cct aga ttg ggt gtg cgc gcg acg agg aag act 198 Leu ProArg Arg Gly Pro Arg Leu Gly Val Arg Ala Thr Arg Lys Thr 40 45 50 tcc gagcgg tcg caa cct cga ggt aga cgt cag cct atc ccc aag gca 246 Ser Glu ArgSer Gln Pro Arg Gly Arg Arg Gln Pro Ile Pro Lys Ala 55 60 65 cgt cgg cccaag ggc agg aac tgg gct cag ccc ggg tat cct tgg ccc 294 Arg Arg Pro LysGly Arg Asn Trp Ala Gln Pro Gly Tyr Pro Trp Pro 70 75 80 ctc tat ggc aatgag ggt tgc ggg tgg gcg gga tgg ctc ctg tcc ccc 342 Leu Tyr Gly Asn GluGly Cys Gly Trp Ala Gly Trp Leu Leu Ser Pro 85 90 95 100 agt ggc tct cggcct agt tgg ggc ccc aac gac ccc cga cgt agg tcg 390 Ser Gly Ser Arg ProSer Trp Gly Pro Asn Asp Pro Arg Arg Arg Ser 105 110 115 cgc aat ttg ggtaag gtc atc gat acc ctt acg tgc ggc ttc gtc gat 438 Arg Asn Leu Gly LysVal Ile Asp Thr Leu Thr Cys Gly Phe Val Asp 120 125 130 ctc atg ggg tacata ccg ctc gtc ggc gcc cct ctt aga ggc gct gcc 486 Leu Met Gly Tyr IlePro Leu Val Gly Ala Pro Leu Arg Gly Ala Ala 135 140 145 agg gcc ctg gcgcat ggc gtc cgg gtt ctg gaa gac ggt gtg aac tat 534 Arg Ala Leu Ala HisGly Val Arg Val Leu Glu Asp Gly Val Asn Tyr 150 155 160 gca aca ggt aacctt cct ggt tgc tct ttc tct atc ttc ctt ctg gcc 582 Ala Thr Gly Asn LeuPro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala 165 170 175 180 ctg ctc tcttgc ctg act gtg ccc gct tca gcc tac caa gtg cgc aac 630 Leu Leu Ser CysLeu Thr Val Pro Ala Ser Ala Tyr Gln Val Arg Asn 185 190 195 2 60 DNAHepatitis C Virus CDS (1)...(60) Corresponds to aa 125 to 144 of SEQ ID.No. 1 2 acc ctt acg tgc ggc ttc gtc gat ctc atg ggg tac ata ccg ctc gtc48 Thr Leu Thr Cys Gly Phe Val Asp Leu Met Gly Tyr Ile Pro Leu Val 1 510 15 ggc gcc cct ctt 60 Gly Ala Pro Leu 20 3 18 DNA Hepatitis C VirusCDS (1)...(18) Corresponds to aa 161-166 of SEQ ID. No. 1 3 ggt gtg aactat gca aca 18 Gly Val Asn Tyr Ala Thr 1 5 4 1900 DNA Human misc_feature(1)...(1900) n = A,T,C or G 4 cgtcttcggg acgcgcccgc tcttcgcctttcgctgcagt ccgtcgattt ctttctccag 60 gaagaaaaat ggcatccgtt gcagttgatccacaaccgag tgtggtgact cgggtggtca 120 acctgccctt ggtgagctcc acgtatgacctcatgtcctc agcctatctc agtacaaagg 180 accagtatcc ctacctgaag tctgtgtgtgagatgscaga gaacggtgtg aagaccatca 240 cctccgtggc catgaccagt gctctgcccatcatccagaa gctagagccg caaattgcag 300 ttgccgatac ctatgcctgt aaggggctagacaggattga ggagagactg cctattctga 360 atcagccatc aactcagatt gttgccaatgccaaaggcgc tgtgactggg gcaaaagatg 420 ctgtgacgac tactgtgact ggggccaaggattctgtngc cagcacgatc acaggggtga 480 tggacaagac caaaggggca gtgactggcagtgtggagaa gaccaagtct gtggtcagtg 540 gcagcattaa cacagtcttg gggagtcggatgatgcagct cgtgagcagt ggcgtagaaa 600 atgcactcac caaatcagag ctgttggtagaacagtacct ccctctcact gaggaagaac 660 tagaaaaaga agcaaaaaaa gttgaaggatttgatctggt tcagaagcca agttattatg 720 ttagactggg atccctgtct accaagcttcactcccgtgc ctaccagcag gctctcagca 780 gggttaaaga agctaagcaa aaaagccaacagaccatttc tcagctccat tctactgttc 840 acctgattga atttgccagg aagaatgtgtatagtgccaa tcagaaaatt caggatgctc 900 aggataagct ctacctctca tgggtagagtggaaaaggag cattggatat gatgatactg 960 atgagtccca ctgtgctgag cacattgagtcacgtactct tgcaattgcc cgcaacctga 1020 ctcagcagct ccagaccacg tgccacaccctcctgtccaa catccaaggt gtaccacaga 1080 acatccaaga tcaagccaag cacatgggggtgatggcagg cgacatctac tcagtgttcc 1140 gcaatgctgc ctcctttaaa gaagtgtctgacagcctcct cacttctagc aaggggcagc 1200 tgcagaaaat gaaggaatct ttagatgacgtgatggatta tcttgttaac aacacgcccc 1260 tcaactggct ggtaggtccc ttttatcctcagctgactga gtctcagaat gctcaggacc 1320 aaggtgcaga gatggacaag agcagccaggagacccagcg atctgagcat aaaactcatt 1380 aaacctgccc ctatcactag tgcatgctgtggccagacag atgacacctt ttgttatgtt 1440 gaaattaact tgctaggcaa ccctaaattgggaagcaagt agctagtata aaggccctca 1500 attgtagttg tttccagctg aattaagagctttaaagttt ctggcattag cagatgattt 1560 ctgttcacct ggtaagaaaa gaatgataggcttgtcagag cctatagcca gaactcagaa 1620 aaaattcaaa tgcacttatg ttctcattctatggccattg tgttgcctct gttactgttt 1680 gtattgaata aaaacatctt catgtgggctggggtagaaa ctggtgtctg ctctggtgtg 1740 atctgaaaag gcgtcttcac tgctttatctcatgatgctt gcttgtaaaa cttgatttta 1800 gtttttcatt tctcaaatag gaatactacctttgaattca ataaaattca ctgcaggata 1860 gaccagttna gnagcaaaca nncangtacacnnaaganac 1900 5 437 PRT Human VARIANT (1)...(437) Xaa = Any Amino Acid5 Met Ala Ser Val Ala Val Asp Pro Gln Pro Ser Val Val Thr Arg Val 1 5 1015 Val Asn Leu Pro Leu Val Ser Ser Thr Tyr Asp Leu Met Ser Ser Ala 20 2530 Tyr Leu Ser Thr Lys Asp Gln Tyr Pro Tyr Leu Lys Ser Val Cys Glu 35 4045 Met Xaa Glu Asn Gly Val Lys Thr Ile Thr Ser Val Ala Met Thr Ser 50 5560 Ala Leu Pro Ile Ile Gln Lys Leu Glu Pro Gln Ile Ala Val Ala Asp 65 7075 80 Thr Tyr Ala Cys Lys Gly Leu Asp Arg Ile Glu Glu Arg Leu Pro Ile 8590 95 Leu Asn Gln Pro Ser Thr Gln Ile Val Ala Asn Ala Lys Gly Ala Val100 105 110 Thr Gly Ala Lys Asp Ala Val Thr Thr Thr Val Thr Gly Ala LysAsp 115 120 125 Ser Val Ala Ser Thr Ile Thr Gly Val Met Asp Lys Thr LysGly Ala 130 135 140 Val Thr Gly Ser Val Glu Lys Thr Lys Ser Val Val SerGly Ser Ile 145 150 155 160 Asn Thr Val Leu Gly Ser Arg Met Met Gln LeuVal Ser Ser Gly Val 165 170 175 Glu Asn Ala Leu Thr Lys Ser Glu Leu LeuVal Glu Gln Tyr Leu Pro 180 185 190 Leu Thr Glu Glu Glu Leu Glu Lys GluAla Lys Lys Val Glu Gly Phe 195 200 205 Asp Leu Val Gln Lys Pro Ser TyrTyr Val Arg Leu Gly Ser Leu Ser 210 215 220 Thr Lys Leu His Ser Arg AlaTyr Gln Gln Ala Leu Ser Arg Val Lys 225 230 235 240 Glu Ala Lys Gln LysSer Gln Gln Thr Ile Ser Gln Leu His Ser Thr 245 250 255 Val His Leu IleGlu Phe Ala Arg Lys Asn Val Tyr Ser Ala Asn Gln 260 265 270 Lys Ile GlnAsp Ala Gln Asp Lys Leu Tyr Leu Ser Trp Val Glu Trp 275 280 285 Lys ArgSer Ile Gly Tyr Asp Asp Thr Asp Glu Ser His Cys Ala Glu 290 295 300 HisIle Glu Ser Arg Thr Leu Ala Ile Ala Arg Asn Leu Thr Gln Gln 305 310 315320 Leu Gln Thr Thr Cys His Thr Leu Leu Ser Asn Ile Gln Gly Val Pro 325330 335 Gln Asn Ile Gln Asp Gln Ala Lys His Met Gly Val Met Ala Gly Asp340 345 350 Ile Tyr Ser Val Phe Arg Asn Ala Ala Ser Phe Lys Glu Val SerAsp 355 360 365 Ser Leu Leu Thr Ser Ser Lys Gly Gln Leu Gln Lys Met LysGlu Ser 370 375 380 Leu Asp Asp Val Met Asp Tyr Leu Val Asn Asn Thr ProLeu Asn Trp 385 390 395 400 Leu Val Gly Pro Phe Tyr Pro Gln Leu Thr GluSer Gln Asn Ala Gln 405 410 415 Asp Gln Gly Ala Glu Met Asp Lys Ser SerGln Glu Thr Gln Arg Ser 420 425 430 Glu His Lys Thr His 435 6 31 PRTArtificial Sequence branched peptide containing residues 5-27 of HCVcore protein 6 Xaa Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Xaa Arg ArgPro Gln 1 5 10 15 Asp Val Lys Phe Pro Gly Gly Lys Lys Lys Lys Lys LysLys Ala 20 25 30 7 11 DNA Artificial Sequence oligonucleotides used toconstruct HCV core protein deletion plasmids 7 gctgagatct a 11 8 29 DNAArtificial Sequence oligonucleotides used to construct HCV core proteindeletion plasmids 8 gtaaccttcc tggttgctct tgagatcta 29 9 17 DNAArtificial Sequence oligonucleotides used to construct HCV core proteindeletion plasmids 9 gtaacctttg agatcta 17 10 18 DNA Artificial Sequenceoligonucleotides used to construct HCV core protein deletion plasmids 10ctggcgcatt gagatcta 18 11 28 DNA Artificial Sequence oligonucleotidesused to construct HCV core protein deletion plasmids 11 ctggcccatggtgttaacta tgcaacag 28 12 31 DNA Artificial Sequence oligonucleotidesused to construct HCV core protein deletion plasmids 12 ctggcccatggcgtccgggt tctggaagac g 31 13 37 DNA Artificial Sequenceoligonucleotides used to construct HCV core protein deletion plasmids 13cgatagaggc gctgccaggg ccctggcgtg agatcta 37 14 52 DNA ArtificialSequence HCV1 oligonucleotide for plasmid construction 14 catggggtacatagcgctcg tcggcgccgc cttaagaggc gctgcgaggg cc 52 15 36 DNA ArtificialSequence HCV2 oligonucleotide for plasmid construction 15 ctagagagcgcaagacgccc cgcgtcaccg gcggcg 36 16 26 DNA Artificial Sequence primerderived from GBV-B, nucleotides 428-448, for plasmid construction 16ggagatctcg tagaccgtag cacatg 26 17 38 DNA Artificial Sequence primerderived from GBV-B, nucleotides 842-868, for plasmid construction 17ggggatccct agtggacacc gaaccaacca gtagccca 38 18 36 DNA ArtificialSequence primer derived from GBV-B, nucleotides 1003-1029, for plasmidconstruction 18 ggggatcctc agatcacaca accaggctcg tgtagg 36 19 27 DNAArtificial Sequence primer derived from GBV-B, nucleotides 1618-1639,for plasmid construction 19 gggtactcta gagtgatagg cctggtc 27 20 49 DNAArtificial Sequence primer derived from GBV-B for plasmid construction20 ctagagagcg caagacgccg cgggtcaccg gtggctctcg caatcttgg 49

What is claimed is:
 1. A method for identifying a substance capable ofaffecting a viral infection, comprising: providing a lipid globuletargeting sequence, as a first component; providing a lipid globule, asa second component; contacting the two components with a substance to betested under conditions that would permit the two components to interactin the absence of the substance; and determining whether the substancedisrupts the interaction between the first and second components.
 2. Themethod of claim 1, wherein the targeting sequence comprises a hepatitisC virus (HCV) core protein or a fragment thereof, or a GB virus-B coreprotein or a fragment thereof.
 3. The method of claim 2, wherein thetargeting sequence further comprises variants or homologues of thehepatitis C virus (HCV) core protein or the GB virus-B core protein. 4.The method according to claim 1, wherein the substance to be tested isadministered to a cell, the lipid globule targeting sequence isexpressed in said cell and the lipid globule is a natural constituent ofsaid cell.
 5. The method according to claim 4, wherein the lipid globuletargeting sequence is naturally or recombinantly expressed in said cell.6. The method according to claim 1, further comprising: administering avirus to a cell in the absence of said substance which has beendetermined to disrupt the interaction between the first and secondcomponents; administering the virus to the cell in the presence of saidsubstance; and determining if said substance reduces or abolishes thesusceptibility of the cell to viral infection or the effects of viralinfection.
 7. The method according to claim 1, wherein the lipid globuletargeting sequence comprises amino acids of the HCV core proteinselected from the group consisting of 125 to 144, 161 to 166 and thecombination thereof.
 8. The method according to claim 1, wherein theviral infection is a hepatitis infection or other viral infection of thehuman or animal liver.
 9. The method according to claim 4, wherein saidcell is a liver cell.
 10. The substance identified by the method ofclaim
 1. 11. The substance according to claim 10, wherein said substancehas not previously been known to affect viral infection.
 12. A methodfor identifying a substance for treating or preventing a viralinfection, comprising: administering said substance to a mammalian cell;and identifying whether the administration of said substance upregulatesexpression of adipocyte-specific differentiation related protein (ADRP)in the mammalian cell.
 13. A substance capable of disrupting aninteraction between a lipid globule targeting sequence and a lipidglobule for use in affecting a viral infection, wherein the targetingsequence comprises a hepatitis C virus (HCV) core protein or a fragmentthereof, or a GB virus-B core protein or fragment thereof.
 14. Apolypeptide comprising a lipid globule targeting sequence for use inpreventing or treating a viral infection, wherein the targeting sequencecomprises an HCV core protein or a fragment, variant or homologuethereof, or a GB virus-B core protein or a fragment, variant orhomologue thereof.
 15. The polypeptide according to claim 14, whereinthe targeting sequence comprises amino acids of the HCV core proteinselected from the group consisting of 125 to 144, 161 to 166 and thecombination thereof.
 16. A pharmaceutical composition comprising thepolypeptide according to claim 15 and a pharmaceutically acceptablecarrier or diluent.
 17. A method for treating or preventing a viralinfection comprising administering an effective amount of thepharmaceutical composition of claim
 16. 18. A method for treating orpreventing a viral infection comprising administering an effectiveamount of a pharmaceutical composition comprising the substanceidentified by claim 1 and a pharmaceutically acceptable carrier ordiluent.
 19. A method for treating or preventing a viral infectioncomprising administering an effective amount of a pharmaceuticalcomposition comprising the substance identified by claim 12 and apharmaceutically acceptable carrier or diluent.
 20. A polynucleotideencoding a polypeptide according to claim 14 for use in treating orpreventing a viral infection.
 21. A method for determining whether atest substance is capable of treating or preventing a viral infection,comprising: providing a lipid globule targeting sequence, as a firstcomponent, said targeting sequence comprising a hepatitis C virus (HCV)core protein or a fragment thereof, or a GB virus-B core protein orfragment thereof; providing a lipid globule, as a second component;incubating the first and second components with the test substance underconditions that would permit the first and second components to interactwith one another in the absence of the test substance; and determiningwhether the test substance disrupts the interaction between the firstand second components.